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WO2002066653A2 - Banques procaryotiques et leurs utilisations - Google Patents

Banques procaryotiques et leurs utilisations Download PDF

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Publication number
WO2002066653A2
WO2002066653A2 PCT/US2001/049058 US0149058W WO02066653A2 WO 2002066653 A2 WO2002066653 A2 WO 2002066653A2 US 0149058 W US0149058 W US 0149058W WO 02066653 A2 WO02066653 A2 WO 02066653A2
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nucleic acid
protein
enzyme
nam
library
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PCT/US2001/049058
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WO2002066653A3 (fr
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Min Li
Yuan-Ching Liu
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Xencor
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1075—Isolating an individual clone by screening libraries by coupling phenotype to genotype, not provided for in other groups of this subclass

Definitions

  • the invention relates to methods and compositions for the construction of prokaryotic libraries expressing candidate proteins and the use of these libraries to identify candidate proteins and the nucleic acids encoding them.
  • Biomolecular display technologies which allow the construction of a large pool of modularly coded biomolecules, their display for property selection, and rapid characterization (decoding) of their structures, are particularly useful for accessing and analyzing protein diversity on a large scale.
  • display technologies have comprised two major groups: biological display systems that employ biological host/biological reactions, and non-biological display systems that use chemical and engineering techniques. Regardless of the format, a display library consists of modularly coded molecules, each of which contains three components: displayed entities, a common linker, and the corresponding individualized codes.
  • display formats Over the past decade, many display formats have been developed and applied in biological and pharmaceutical research. These technologies use different types of displayed entities, linkage formats, and coding strategies.
  • Display libraries vary widely in size and complexity. On the basis of theoretical calculation as well as experimental results, these parameters essentially determine the probability and quality of identified biomolecules (Perelson and Oster, (1979) J. Theor. Biol., 81 :645-670). Although increasing both the size and the complexity of a display library is an important objective, the fundamental aim is to optimize the assembly of building blocks that potentially lead to novel and more diverse properties.
  • Bio display exploits the cellular biosynthesis machinery to assemble biopolymers, the sequence of which ultimately specifies structure and distinct properties.
  • nucleotide polymers such as RNA/DNA aptamers
  • RNA/DNA aptamers have yielded interesting molecules (Patel and Suir, (2000) J. Biotechnol. 74:39- 60)
  • the most commonly exploited for biological display is the nucleic acid coded synthesis of L-amino acid polymers (i.e., proteins).
  • Most biological display systems use the 20 natural L-amino acids as building blocks and take advantage of enzymatic protein synthesis.
  • One of the most important characteristics of display technologies is the ability to determine the structure of a desired compound rapidly after initial screening. Structural or sequence characterization is often accomplished by a process commonly known as coding and decoding, which can be achieved via a coupled amplification and purification process.
  • chemical entities are linked to codes that have chemical and physical properties that can be readily determined, such as the sequence of nucleic acids (Brenner, S. and Lerner, R.A. (1992) Proc. Natl. Acad. Sci. USA, 89:5381- 5383).
  • a linker is used to establish the modularly coded biomolecular units, each of which possesses a unique property for either detection or deconvolution because of the attached codes. In most cases, the linkage between the displayed entity and the corresponding code is achieved by physical connections via either covalent or non-covalent chemical binding.
  • coding formats Three types of coding formats are commonly used: peptide-on-DNA/RNA display, viral (phage) display, and cell-based display.
  • the first of these formats uses protein-DNA/RNA complexes as its foundation. By expressing the peptide in a form that is capable of binding to its coding DNA/RNA, one can screen a large pool of complexes and identify bound peptides by the isolation and sequencing of nucleotide sequences of either DNA or RNA.
  • the second type of display format is one of the most commonly used (Smith, G.P, (1985) Science, 228:1315-1317; Dulbecco, R., U.S. Patent No. 4,593,002; Ladner, R. ., et al., U.S. Patent No. 5,837,500; Ladner, R.C. et al., U.S. Patent No. 5,223,409; Dower, et al., U.S. Patent No. 5,427,908; Russell et al., U.S. Patent No. 5,723,287; Li U.S. Patent No. 6,190,856).
  • a cell-based display can be used to display large cDNA libraries in mammalian cells.
  • This system requires efficient gene transfer, such as through the use of a viral vector.
  • the cellular host is used to establish the link between the coding DNA and the displayed peptides/proteins.
  • Plasmid display is an alternative approach to the display methods described above and avoids potential difficulties with secretion, in vitro translation, or RNA stability by fusing polypeptides directly to a DNA binding protein (Cull, et al., (1992) Proc. Natl. Acad. Sci., USA 89:1865-1869; Speight, et al., (2001) Chemistry and Biology, 8:951-965).
  • the present invention provides methods for generating libraries of fusing nucleic acids by providing procaryotic expression vectors comprising a T7 promoter operably linked to a nucleic acid encoding a nucleic acid modification enzyme (NAM), a candidate protein and an enzyme attachment sequence (EAS) that is recognized by the NAM enzyme.
  • NAM nucleic acid modification enzyme
  • EAS enzyme attachment sequence
  • the invention provides libraries of nucleic acid/protein (NAP) conjugates each comprising a fusion polypeptide comprising a NAM enzyme and a candidate protein.
  • NAP conjugates also comprise a procaryotic expression vector comprising a T7 promoter operably linked to a nucleic acid encoding a nucleic acid modification enzyme (NAM), a candidate protein and an enzyme attachment sequence (EAS) that is recognized by the NAM enzyme.
  • NAM nucleic acid modification enzyme
  • EAS enzyme attachment sequence
  • the invention provides libraries of procaryotic host cells each comprising a fusion polypeptide comprising a NAM enzyme and a candidate protein.
  • the NAP conjugates also comprise a procaryotic expression vector comprising a T7 promoter operably linked to a nucleic acid encoding a nucleic acid modification enzyme (NAM), a candidate protein and an enzyme attachment sequence (EAS) that is recognized by the NAM enzyme.
  • NAM nucleic acid modification enzyme
  • EAS enzyme attachment sequence
  • the invention provides a library of expression vectors comprising a nucleic acid under the control of an expressible promoter wherein said nucleic acid encodes a NAM enzyme, an EAS that is recognized by said NAM enzyme and a nucleic acid encoding a transposon sequence.
  • the invention provides libraries of procaryotic host cells each comprising a fusion nucleic acid comprising a nucleic acid under the control of an expressible promoter wherein said nucleic acid encodes a NAM enzyme, a nucleic acid encoding a candidate host cell protein, an EAS that is recognized by said NAM enzyme and a nucleic acid encoding a transposon sequence and a fusion polypeptide comprising a NAM enzyme and a candidate host cell protein.
  • the invention provides methods of screening comprising expressing a library of expression vectors in a suitable procaryotic host cell under condition wherein a library of NAP conjugates is formed, adding at least one target molecule, and determining the binding of a NAP conjugate to the target.
  • the invention provides methods of screening comprising expressing a library of expression vectors in a suitable procaryotic host cell under condition wherein a library of NAP conjugates is formed, and screening the host cells for an altered phenotype.
  • Procaryotic expression vectors of use in the present invention include pET-24a and GatewayTM vectors.
  • Suitable NAM enzymes include Rep 68, Rep 78 and variants of Rep 68.
  • EASs comprise at least 50 nucleotides, although EASs up to 165 nucleotides may be used in the methods of the present invention.
  • Figure 1 depicts the amino acid sequence of Rep78 isolated from adeno-associated virus 2.
  • Figure 2 depicts the nucleotide sequence of Rep78 isolated from adeno-associated virus 2.
  • Figure 3 depicts the amino acid sequence of major coat protein A isolated from adeno- associated virus 2.
  • Figure 4 depicts the nucleotide sequence of major coat protein A isolated from adeno- associated virus 2.
  • Figure 5 depicts the amino acid sequence of a Rep protein isolated from adeno- associated virus 4.
  • Figure 6 depicts the nucleotide sequence of a Rep protein isolated from adeno- associated virus 4.
  • Figure 7 depicts the amino acid sequence of Rep78 isolated from adeno-associated virus 3B.
  • Figure 8 depicts the nucleotide sequence of Rep78 isolated from adeno-associated virus 3B.
  • Figure 9 depicts the amino acid sequence of a nonstructural protein isolated from adeno-associated virus 3.
  • Figure 10 depicts the nucleotide sequence of a nonstructural protein isolated from adeno-associated virus 3.
  • Figure 11 depicts the amino acid sequence of a nonstructural protein isolated from adeno-associated virus 1.
  • Figure 12 depicts the nucleotide sequence of a nonstructural protein isolated from adeno-associated virus 1.
  • Figure 13 depicts the amino acid sequence of Rep78 isolated from adeno-associated virus 6.
  • Figure 14 depicts the nucleotide sequence of Rep78 isolated from adeno-associated virus 6.
  • Figure 15 depicts the amino acid sequence of Rep68 isolated from adeno-associated virus 2.
  • Figure 16 depicts the nucleotide sequence of Rep68 isolated from adeno-associated virus 2.
  • Figure 17 depicts the amino acid sequence of major coat protein A' (alt.) isolated from adeno-associated virus 2.
  • Figure 18 depicts the nucleotide sequence of major coat protein A' (alt.) isolated from adeno-associated virus 2.
  • Figure 19 depicts the amino acid sequence of major coat protein A" (alt.) isolated from adeno-associated virus 2.
  • Figure 20 depicts the nucleotide sequence of major coat protein A" (alt.) isolated from adeno-associated virus 2.
  • Figure 21 depicts the amino acid sequence of a Rep protein isolated from adeno- associated virus 5.
  • Figure 22 depicts the nucleotide sequence of a Rep protein isolated from adeno- associated virus 5.
  • Figure 23 depicts the amino acid sequence of major coat protein Aa (alt.) isolated from adeno-associated virus 2.
  • Figure 24 depicts the nucleotide sequence of major coat protein Aa (alt.) isolated from adeno-associated virus 2.
  • Figure 25 depicts the amino acid sequence of a Rep protein isolated from Barbarie duck parvovirus.
  • Figure 26 depicts the nucleotide sequence of a Rep protein isolated from Barbarie duck parvovirus.
  • Figure 27 depicts the amino acid sequence of a Rep protein isolated from goose parvovirus.
  • Figure 28 depicts the nucleotide sequence of a Rep protein isolated from goose parvovirus.
  • Figure 29 depicts the amino acid sequence of NS1 isolated from muscovy duck parvovirus.
  • Figure 30 depicts the nucleotide sequence of NS1 isolated from muscovy duck parvovirus.
  • Figure 31 depicts the amino acid sequence of NS1 isolated from goose parvovirus.
  • Figure 32 depicts the nucleotide sequence of NS1 isolated from goose parvovirus.
  • Figure 33 depicts the amino acid sequence of non-structural protein 1 isolated from chipmunk parvovirus.
  • Figure 34 depicts the nucleotide sequence of non-structural protein 1 isolated from chipmunk parvovirus.
  • Figure 35 depicts the amino acid sequence of non-structural protein isolated from the pig-tailed macaque parvovirus.
  • Figure 36 depicts the nucleotide sequence of non-structural protein isolated from the pig-tailed macaque parvovirus.
  • Figure 37 depicts the amino acid sequence of NS1 isolated from a simian parvovirus.
  • Figure 38 depicts the nucleotide sequence of NS1 protein isolated from a simian parvovirus.
  • Figure 39 depicts the amino acid sequence of a NS protein isolated from the Rhesus macaque parvovirus.
  • Figure 40 depicts the nucleotide sequence of a NS protein isolated from the Rhesus macaque parvovirus.
  • Figure 41 depicts the amino acid sequence of a non-structural protein isolated from the B19 virus.
  • Figure 42 depicts the nucleotide sequence of a non-structural protein isolated from the B19 virus.
  • Figure 43 depicts the amino acid sequence of orf 1 isolated from the Erythrovirus B19.
  • Figure 44 depicts the nucleotide sequence of the product of orf 1 isolated from the Erythrovirus B19.
  • Figure 45 depicts the amino acid sequence of U94 isolated from the human herpesvirus 6B.
  • Figure 46 depicts the nucleotide sequence of U94 isolated from the human herpesvirus 6B.
  • Figure 47 depicts an enzyme attachment site for a Rep protein.
  • Figure 48 depicts the Rep 68 and Rep 78 enzyme attachment site found in chromosome 19.
  • Figure 49A depicts the nucleotide sequence of the Rep 68 variant, PCD 302.
  • Figure 49B depicts the amino acid sequence of the Rep 68 variant, PCD302.
  • Figures 50A-D depict the nucleotide sequence of enzyme attachment sites (EAS) of use in the invention.
  • Figure 50 A depicts an EAS found within the inverted terminal repeat sequence of adeno- associated virus 2 (SEQ ID NO:57).
  • Figure 50B depicts the nucleotide sequence of a 165 base pair EAS (SEQ ID NO:58). Highlighted sequences represent two flanking restriction enzyme sites used for cloning
  • Figure 50C depicts the sequence of a 80 base pair EAS (SEQ ID NO 59)
  • Figure 50 D depicts the sequence of a 50 base pair EAS (SEQ ID NO 60) Base pairs appearing in bold represent flanking restriction enzyme sites used for cloning
  • Figure 51 depicts an example of an RNA-protein fusion of use in the methods of the present invention
  • Figure 52 depicts a transposon based expression vector based on a Tn5 mini-transposon comprising an origin of replication for R6K, a NAM enzyme under the control of a promoter (Pr), an EAS, an antibiotic marker (i e , kanamycin), and nucleic acid sequences encoding the inside (I) and outside (O) edge of Tn5
  • Figure 53 depicts the pQE82L-based plasmid expression vector into which the nucleic acid sequences encoding PCD302 and the 165 bp EAS were cloned
  • Figure 54 depicts the pET-24-a(+) based plasmid expression used for the cloning of nucleic acid sequences encoding NAM/candidate protein gene fusions and EASs
  • Figure 55 depicts the insertion of PCD302 into the Hindlll site of pET-24a(+)
  • Figure 56 depicts the pBAD based plasmid expression Fusion nucleic acid sequences encoding NAM/candidate protein fusions are inserted into the multiple cloning site (MCS), such that expression of the NAM enzyme is under the control of the PBAD promoter
  • Figures 57A and 57B depicts pQE82L based plasmid constructs comprising PCD302/MBP ( Figure 58A) or PCD302/FKBP ( Figure 58B) fusions
  • Figure 58 depicts the sequence of a the PCD302/MBP fusion nucleic acid (SEQ ID NO 62)
  • the first arrow indicates the beginning of the nucleic acid sequence encoding PCD302
  • the second arrow indicates the end of the PCD302 nucleic acid sequence
  • the third arrow indicates the beginning of the MalE nucleic acid sequence (with the signal sequence deleted)
  • the last arrow indicates the end of the MalE sequence
  • Figure 59A depicts a pQE82L-based GatewayTM compatible plasmid vector in which the Hindlll site was mutated to SnaBI
  • the nucleotide sequence of the Gateway transfer cassette (shown in the insert) is shown in Figure 59B (SEQ ID NO 63)
  • Figure 60A depicts the results from a DNA agarose gel used to analyze host cells transformed with PCD302/MBP.
  • Figure 61 depicts the results from a western blot analysis to determine the number of colonies carrying a PCD302/candidate fusions in the proper orientation and reading frame.
  • Figures 62 and 63 depict the level of sensitivity at which cells transformed with NAM/candidate protein gene fusions can be detected using the screening protocols described herein.
  • the present invention is directed to a novel method that can allow the rapid and facile identification of these "hits".
  • the disclosed method is conceptually distinct from prior display technologies based on plasmid display in that it relies on the use of nucleic acid modification enzymes that covalently and specifically bind to the nucleic acid molecules comprising the sequence that encodes them.
  • Proteins of interest for example, candidates to be screened either for binding to disease-related proteins or for a phenotypic effect
  • NAM nucleic acid modification
  • the NAM enzyme will covalently attach itself to a corresponding NAM attachment sequence (termed an enzyme attachment sequence (EAS)).
  • the candidate protein is covalently linked to the nucleic acid that encodes it upon translation.
  • candidates that exhibit the desired properties can be quickly isolated using a variety of methods such as PCR amplification. This facilitates the quick identification of useful candidate proteins, and allows rapid screening and validation to occur.
  • nucleic acid or “oligonucleotide” or grammatical equivalents herein means at least two nucleosides covalently linked together.
  • a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases nucleic acid analogs are included that may have alternate backbones, particularly when probes are used, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem.
  • nucleic acid analogs may find use in the present invention.
  • mixtures of naturally occurring nucleic acids and analogs can be made, or, alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
  • the nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo- nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
  • nucleoside includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides.
  • nucleoside includes non-naturally occurring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
  • fusion nucleic acid herein is meant a plurality of nucleic acid components (e.g., peptide coding sequences) that are joined together.
  • the fusion nucleic acids preferably encode fusion polypeptides, although this is not required.
  • fusion polypeptide or “fusion peptide” or grammatical equivalents herein is meant a protein composed of a plurality of protein components, that while typically unjoined in their native state, are joined by their respective amino and/or carboxyl termini through a peptide linkage to form a single continuous polypeptide.
  • Plurality in this context means at least two, and preferred embodiments generally utilize two components.
  • the protein components can be joined directly or joined through a peptide linker/spacer as outlined below.
  • the fusion nucleic acids can encode protein components that are not fused; although generally the nucleic acids encoding each component are fused.
  • additional components such as fusion partners including targeting sequences, etc., can be used.
  • the fusion nucleic acids encode nucleic acid modification (NAM) enzymes and candidate proteins.
  • nucleic acid modification enzyme or “NAM enzyme” herein is meant an enzyme that utilizes nucleic acids, particularly DNA, as a substrate and covalently attaches itself to nucleic acid enzyme attachment (EA) sequences. The covalent attachment can be to the base, to the ribose moiety or to the phosphate moieties.
  • NAM enzymes include, but are not limited to, helicases, topoisomerases, polymerases, gyrases, recombinases, transposases, restriction enzymes and nucleases. As outlined below, NAM enzymes include natural and non-natural variants.
  • DNA binding peptides are known, such as those involved in nucleic acid compaction, transcription regulators, and the like, enzymes that covalently attach to nucleic acids, i.e., DNA, in particular peptides involved with replication, are preferred.
  • Some NAM enzymes can form covalent linkages with DNA without nicking the DNA.
  • enzymes involved in DNA repair recognize and covalently attach to nucleic acid regions, which can be either double-stranded or single-stranded.
  • Such NAM enzymes are suitable for use in the fusion enzyme library.
  • DNA NAM enzymes that nick DNA to form a covalent linkage e.g., viral replication peptides, are most preferred.
  • the NAM enzyme is a protein that recognizes specific sequences or conformations of a nucleic acid substrate and performs its enzymatic activity such that a covalent complex is formed with the nucleic acid substrate
  • the enzyme acts upon nucleic acids, particularly DNA, in various configurations including, but not limited tor single-strand DNA, double-strand DNA, Z-form
  • Suitable NAM enzymes include, but are not limited to, enzymes involved in replication such as Rep68 and Rep78 of adeno-associated viruses (AAV), NS1 and H-1 of parvovirus, bacteriophage ph ⁇ -29 terminal proteins, the 55 Kd adenovirus proteins, and derivatives thereof
  • the NAM enzyme is a Rep protein Rep proteins include, but are not limited to, Rep78, Rep68, and functional homologues thereof found in related viruses Rep proteins, including their functional homologues, may be isolated from a variety of sources including parvoviruses, erythroviruses, herpesviruses, and other related viruses
  • Rep proteins may be isolated from a variety of sources including parvoviruses, erythroviruses, herpesviruses, and other related viruses
  • the natural Rep protein can be mutated or engineered with techniques known in the art in order to improve its activity or reduce its potential toxicity Such experimental improvements may done in conjunction with native or variants of their corresponding EAS
  • One of preferred Rep proteins is the AAV Rep protein Adeno-associated viral (AAV) Rep proteins are encoded by the left open reading frame of the viral genome AAV Rep proteins, such as Rep68 and Rep78, regulate AAV transcription, activate AAV replication, and have been shown to inhibit transcription of heterologous promoters (Chionni et al ,
  • the NAM enzyme is NS1 NS1 is a non-structural protein in parvovirus, is a functional homologue of Rep78, and also covalently attaches to DNA (Cotmore et al , J Virol , 62(3), 851-860 (1998), hereby expressly incorporated by reference)
  • the nucleotide and ammo acid sequences of NS1 proteins isolated from various sources are shown in Figures 9-12, 29-34, 37, and 38 (SEQ ID NOS:9-12, 29-34, 37 & 38).
  • fragments and variants of NS1 proteins are also included within the definition of NS1 proteins.
  • the NAM enzyme is the parvoviral H-1 protein, which is also known to form a covalent linkage with DNA (see, for example, Tseng et al., Proc. Natl. Acad. Sci. USA, 76(11), 5539- 5543 (1979), hereby expressly incorporated by reference.
  • fragments and variants of H-1 proteins are also included within the definition of H-1 proteins.
  • the NAM enzyme is the bacteriophage phi-29 terminal protein, which is also known to form a covalent linkage with DNA (see, for example, Germendia et al., Nucleic Acid Research, 16(3), 5727-5740 (1988), hereby expressly incorporated by reference). As is further outlined below, fragments and variants of phi-29 proteins are also included within the definition of phi- 29 proteins.
  • the NAM enzyme also can be the adenoviral 55 Kd (a55) protein, again known to form covalent linkages with DNA; see Desiderio and Kelly, J. Mol. Biol., 98, 319-337 (1981), hereby expressly incorporated by reference. As is further outlined below, fragments and variants of a55 proteins are also included within the definition of a55 proteins.
  • DNA-binding enzymes form covalent linkages upon physical or chemical stimuli such as, for example, UV-induced cross-linking between DNA and a bound protein, or camptothecin (CPT)-related chemically induced trapping of the DNA-topoisomerase I covalent complex (e.g., Hertzberg et al., J. Biol. Chem., 265, 19287-19295 (1990)).
  • CPT camptothecin
  • NAM enzymes of the present invention are amino acid sequence variants retaining biological activity (e.g., the ability to covalently attach to nucleic acid molecules). These variants fall into one or more of three classes: substitutional, insertional or deletional (e.g. fragment) variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the NAM protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the recombinant DNA in cell culture as outlined herein. However, variant NAM protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis or peptide ligation using established techniques.
  • Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the NAM protein amino acid sequence.
  • the variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
  • the mutation per se need not be predetermined.
  • random mutagenesis may be conducted at the target codon or region and the expressed NAM variants screened for the optimal combination of desired activity.
  • Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants, variants, homologues, etc., is accomplished using assays of NAM protein activities employing routine methods such as, for example, binding assays, affinity assays, peptide conformation mapping, and the like.
  • Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger, for example when unnecessary domains are removed.
  • substitutions deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the NAM protein are desired, substitutions are generally made in accordance with the following chart:
  • substitutions that are less conservative than those shown in Chart I.
  • substitutions may be made which more significantly affect the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • leucyl isoleucyl, phenylalanyl, valyl or alanyl
  • a cysteine or proline is substituted for (or by) any other residue
  • a residue having an electropositive side chain e.g. lysyl, arginyl, or histidyl
  • an electronegative residue e.g. glutamyl or aspartyl
  • a residue having a bulky side chain e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
  • the variants typically exhibit the same qualitative biological activity as the naturally-occurring analogue, although variants also are selected to modify the characteristics of the NAM proteins as needed.
  • the variant may be designed such that the biological activity of the NAM protein is altered. For example, reduced toxicity in the host cell, increased solubility, or increased specificity for the wild-type or variant EAS sequences. Other modification may include alteration or removal of glycosylation sites.
  • functional mutations within the endonuclease domain or nucleic acid recognition site may be made.
  • unnecessary domains may be deleted, to form fragments of NAM enzymes.
  • the NAM is a variant of Rep 68.
  • variant of Rep 68 herein is meant a variant falling into one or more of three classes: substitutional, insertional, or deletional.
  • NAM variants retain sufficient NAM enzymatic activity to find use in screens.
  • the Rep 68 variant is a variant of the wild-type Rep 68 from adeno- associated virus 2; the amino acid and nucleotide sequences are set forth in Figures 15 and 16 (SEQ ID NOS:15 AND 16).
  • the Rep 68 variant is a deletion variant, referred to herein as PCD302, having the nucleotide sequence set forth Figure 49 (SEQ ID NO:55).
  • PCD302 is an EcoRV-Hindlll fragment of wild-type Rep 68 that is more soluble than wild-type Rep 68 in procaryotic host cells. Essentially, PCD302 is missing the first 6 base pairs (atgccg) and the last 42 base pairs from the wild- type Rep 68 sequence shown in Figure 16 (SEQ ID NO:16).
  • some embodiments utilize concatameric constructs to effect multivalency and increase binding kinetics or efficiency.
  • constructs containing a plurality of NAM coding regions or a plurality of EASs may be made.
  • NAM protein also included with the definition of NAM protein are other NAM homologues, and NAM proteins from other organisms including viruses, which are cloned and expressed as known in the art.
  • probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related NAM proteins.
  • particularly useful probe and/or PCR primer sequences include the unique areas of the NAM nucleic acid sequence.
  • preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed.
  • the conditions for the PCR reaction are well known in the art.
  • the fusion nucleic acids of the invention also encode candidate proteins.
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures, i.e., "analogs” such as peptoids [see Simon et al., Proc. Natl. Acd. Sci. U.S.A. 89(20:9367-71 (1992)], generally depending on the method of synthesis.
  • amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline, and noreleucine are considered amino acids for the purposes of the invention.
  • amino acid also includes imino acid residues such as proline and hydroxyproline.
  • any amino acid representing a component of the variant proteins of the present invention can be replaced by the same amino acid but of the opposite chirality.
  • any amino acid naturally occurring in the L-configuration (which may also be referred to as the R or S, depending upon the structure of the chemical entity) may be replaced with an amino acid of the same chemical structural type, but of the opposite chirality, generally referred to as the D- amino acid but which can additionally be referred to as the R- or the S-, depending upon its composition and chemical configuration.
  • Such derivatives have the property of greatly increased stability, and therefore are advantageous in the formulation of compounds which may have longer in vivo half lives, when administered by oral, intravenous, intramuscular, intraperitoneal, topical, rectal, intraocular, or other routes.
  • the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations. Proteins including non-naturally occurring amino acids may be synthesized or in some cases, made recombinantly; see van Hest et al., FEBS Lett 428:(1-2) 68-70 May 22 1998 and Tang et al., Abstr. Pap Am. Chem. S218:U138-U138 Part 2 August 22, 1999, both of which are expressly incorporated by reference herein.
  • the candidate proteins of the present invention may be from prokaryotes and eukaryotes, such as bacteria (including extremeophiles such as the archebacteria), fungi, insects, fish, and mammals.
  • Suitable mammals include, but are not limited to, rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc) and in the most preferred embodiment, from humans.
  • the nucleic acid sequences may be naturally occurring nucleic acids, such as cDNAs or genomic DNAs, random nucleic acids, or "biased" random nucleic acids.
  • Suitable candidate proteins include, but are not limited to, industrial, pharmaceutical, and agricultural proteins, including ligands, cell surface receptors, antigens, antibodies, cytokines, hormones, transcription factors, signaling modules, cell cycle check point proteins, cytoskeletal proteins and enzymes.
  • Suitable classes of enzymes include, but are not limited to, hydrolases such as proteases, carbohydrases, lipases; isomerases such as racemases, epimerases, tautomerases, or mutases; transferases, kinases, oxidoreductases, and phophatases.
  • hydrolases such as proteases, carbohydrases, lipases
  • isomerases such as racemases, epimerases, tautomerases, or mutases
  • transferases kinases, oxidoreductases, and phophatases.
  • Suitable enzymes are listed in the Swiss-Prot enzyme database.
  • Suitable protein backbones include, but are not limited to, all of those found in the protein data base compiled and serviced by the Research Collaboratory for Structural Bioinformatics (RCSB, formerly the Brookhaven National Lab).
  • preferred pharmaceutical candidate proteins include, but are not limited to, those with known structures (including variants) including cytokines (IL-1ra (+receptor complex), IL-1 (receptor alone), IL-1a, IL-1 b (including variants and or receptor complex), IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IFN- ⁇ , INF-Y, IFN- ⁇ -2a; IFN- ⁇ -2B, TNF- ⁇ ; CD40 ligand (chk), Human Obesity Protein Leptin, Granulocyte Colony-Stimulating Factor, Bone Morphogenetic Protein-7, Ciliary Neurotrophic Factor, Granulocyte-Macrophage Colony-Stimulating Factor, Monocyte Chemoattractant Protein 1 , Macrophage Migration Inhibitory Factor, Human Glycosylation-lnhibiting Factor, Human Rantes, Human Macrophage Inflammatory Protein 1 Beta, human growth hormone, Leukemia Infection,
  • preferred industrial candidate proteins include, but are not limited to, those with known structures (including variants) including proteases, (including, but not limited to papains, subtilisins), cellulases (including , but not limited to, endoglucanases I, II, and III, exoglucanases, xylanases, ligninases, cellobiohydrolases I, II, and III, carbohydrases (including, but not limited to glucoamylases, ⁇ -amylases, glucose isomerases) and lipases.
  • proteases including, but not limited to papains, subtilisins
  • cellulases including , but not limited to, endoglucanases I, II, and III, exoglucanases, xylanases, ligninases, cellobiohydrolases I, II, and III
  • carbohydrases including, but not limited to glucoamylases, ⁇ -amylases, glucose isome
  • preferred agricultural candidate proteins include, but are not limited to, those with known structures (including variants) including xylose isomerase, pectinases, cellulases, peroxidases, rubisco, ADP glucose phrophosphorlyase, as well as enzymes involved in oil biosynthesis, sterol biosynthesis, carbohydrate biosynthesis, and the synthesis of secondary metabolites.
  • candidate protein herein is meant a protein to be tested for binding, association or effect in an assay of the invention, including both in vitro (e.g. cell free systems) or ex vivo (within cells).
  • the candidate peptide comprises at least one desired target property.
  • the desired target property will depend upon the particular embodiment of the present invention.
  • Target property refers to an activity of interest.
  • the target property is used directly or indirectly to identify a subset of fusion protein-expression vector conjugates, thus allowing for the retrieval of the desired NAP conjugates from the fusion protein library.
  • Target properties include, for example, the ability of the encoded display peptide to mediate binding to a partner, enzymatic activity, the ability to mimic a given factor, the ability to alter cell physiology, and structural or other physical properties including, but not limited to, electromagnetic behavior or spectroscopic behavior of the peptides.
  • libraries of candidate proteins are used in the fusions.
  • the source of the candidate protein libraries can vary, particularly depending on the end use of the system.
  • the candidate proteins are derived from cDNA libraries.
  • the cDNA libraries can be derived from any number of different cells, particularly those outlined for host cells herein, and include cDNA libraries generated from eucaryotic and procaryotic cells, viruses, cells infected with viruses or other pathogens, genetically altered cells, etc. Preferred embodiments, as outlined below, include cDNA libraries made from different individuals, such as different patients, particularly human patients.
  • the cDNA libraries may be complete libraries or partial libraries.
  • the library of candidate proteins can be derived from a single cDNA source or multiple sources; that is, cDNA from multiple cell types or multiple individuals or multiple pathogens can be combined in a screen.
  • the cDNA library may utilize entire cDNA constructs or fractionated constructs, including random or targeted fractionation. Suitable fractionation techniques include enzymatic, chemical or mechanical fractionation.
  • the candidate proteins are derived from genomic libraries.
  • the genomic libraries can be derived from any number of different cells, particularly those outlined for host cells herein, and include genomic libraries generated from eucaryotic and procaryotic cells, viruses, cells infected with viruses or other pathogens, genetically altered cells, etc.
  • the genomic libraries may be complete libraries or partial libraries.
  • the library of candidate proteins can be derived from a single genomic source or multiple sources; that is, genomic DNA from multiple cell types or multiple individuals or multiple pathogens can be combined in a screen.
  • the genomic library may utilize entire genomic constructs or fractionated constructs, including random or targeted fractionation. Suitable fractionation techniques include enzymatic, chemical or mechanical fractionation.
  • the present invention further provides an isolated and purified nucleic acid molecule comprising a nucleic acid sequence encoding a NAM enzyme fused to a nucleic acid sequence isolated or derived from genomic DNA (for example, vectors comprising genomic digests can be made, or specific genomic sequences can be amplified and/or purified and the amplicons used).
  • an isolated and purified nucleic acid molecule is particularly useful in the present inventive methods described herein.
  • the isolated and purified nucleic acid molecule further comprises a splice donor sequence or splice acceptor sequence located between the nucleic acid sequence encoding the NAM enzyme and the genomic DNA.
  • a splice donor sequence or splice acceptor sequence located between the nucleic acid sequence encoding the NAM enzyme and the genomic DNA.
  • the incorporation of splice donor and/or splice acceptor sequences into the isolated and purified nucleic acid sequence allows formation of a transcript encoding the NAM enzyme and exons of the genomic DNA fragment.
  • the methods of the prior art have failed to comprehend the potential of operably linking genomic DNA to a NAM enzyme such that the product of the genomic DNA can be associated with the nucleic acid molecule encoding it.
  • appropriate regulatory sequences can also be incorporated into the isolated and purified nucleic acid molecule.
  • the present invention also provides methods of determining open reading frames in genomic DNA.
  • the candidate protein encoded by the genomic nucleic acid is preferably fused directly to the N-terminus of the NAM enzyme, rather than at the C-terminus. Thus, if a functional NAM enzyme is produced, the genomic DNA was fused in the correct reading frame. This is particularly useful with the use of labels, as well.
  • the candidate protein library is a random or biased random peptide library. Generally, peptides ranging from about 4 amino acids in length to about 100 amino acids may be used, with peptides ranging from about 5 to about 50 being preferred, with from about 8 to about 30 being particularly preferred and from about 10 to about 25 being especially preferred.
  • libraries may also be subsequently mutated using known techniques (exposure to mutagens, error-prone PCR, error-prone transcription, combinatorial splicing (e.g. cre-lox recombination)).
  • libraries of procaryotic and eukaryotic proteins may be made for screening in the systems described herein.
  • Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, plant, and animal (e.g., mammalian) proteins, with the latter being preferred, and human proteins being especially preferred.
  • the candidate proteins may vary in size. In the case of cDNA or genomic libraries, the proteins may range from 20 or 30 amino acids to thousands, with from about 50 to 1000 (e.g., 75, 150, 350, 750 or more) being preferred and from 100 to 500 (e.g., 200, 300, or 400) being especially preferred.
  • the candidate proteins are peptides, the peptides are from about 3 to about 50 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred.
  • the peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides.
  • each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position.
  • the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
  • libraries of candidate proteins are fused to the NAM enzymes, with each member of the library comprising a different candidate protein.
  • each member of the library may be reproduced or duplicated, resulting in some libraries members being identical.
  • the library should provide a sufficiently structurally diverse population of expression products to effect a probabilistically sufficient range of cellular responses to provide one or more cells exhibiting a desired response. Accordingly, an interaction library must be large enough so that at least one of its members will have a structure that gives it affinity for some molecule, including both protein and non-protein targets, or other factors whose activity is necessary or effective within the assay of interest.
  • the library is fully randomized, with no sequence preferences or constants at any position
  • the library is biased That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities
  • the nucleotides or ammo acid residues are randomized within a defined class, for example, of hydrophobic ammo acids, hydrophilic residues, stencally biased (either small or large) residues, towards the creation of cystemes, for cross-linking, pralines for SH-3 domains, PDZ domains, se ⁇ nes, threonines, tyrosmes or histidmes for phosphorylation sites, etc , or to purines, etc
  • the bias is towards peptides or nucleic acids that interact with known classes of molecules
  • the candidate protein is a peptide
  • a short region from the HIV-1 envelope cytoplasmic domain has been previously shown to block the action of cellular calmodulm Regions of the Fas
  • 13 - cytoplasmic domain which show homology to the mastoparan toxin from Wasps, can be limited to a short peptide region with death-inducing apoptotic or G protein inducing functions.
  • Magainin a natural peptide derived from Xenopus, can have potent anti-tumour and anti-microbial activity.
  • Short peptide fragments of a protein kinase C isozyme ( ⁇ PKC) have been shown to block nuclear translocation of ⁇ PKC in Xenopus oocytes following stimulation.
  • ⁇ PKC protein kinase C isozyme
  • SH-3 target peptides have been used as pseudosubstrates for specific binding to SH-3 proteins.
  • a number of molecules or protein domains are suitable as starting points for the generation of biased randomized candidate proteins.
  • a large number of small molecule domains are known that confer a common function, structure or affinity.
  • areas of weak amino acid homology may have strong structural homology.
  • a number of these molecules, domains, and/or corresponding consensus sequences are known, including, but are not limited to, SH-2 domains, SH-3 domains, Pleckstrin, death domains, protease cleavage/recognition sites, enzyme inhibitors, enzyme substrates, Traf, etc.
  • nucleic acid binding proteins containing domains suitable for use in the invention. For example, leucine zipper consensus sequences are known.
  • biased SH-3 domain-binding oligonucleotides/peptides are made.
  • SH-3 domains have been shown to recognize short target motifs (SH-3 domain-binding peptides), about ten to twelve residues in a linear sequence that can be encoded as short peptides with high affinity for the target SH-3 domain. Consensus sequences for SH-3 domain binding proteins have been proposed.
  • oligos/peptides are made with the following biases:
  • Pro hyd hyd Gly Gly Pro Pro STOP (SEQ ID NO:49) atg ggc nnk nnk nnk nnk nnk aga cct ctg cct cca sbk ggg sbk sbk gga ggc cca cct TAA1 (SEQ ID NO:50).
  • the N-terminus flanking region is suggested to have the greatest effects on binding affinity and is therefore entirely randomized.
  • "Hyd” indicates a bias toward a hydrophobic residue, i.e.- Val, Ala, Gly, Leu, Pro, Arg.
  • the candidate protein is a structural tag that will allow the isolation of target proteins with that structure. That is, in the case of leucine zippers, the fusion of the NAM enzyme to a leucine zipper sequence will allow the fusions to "zip up" with other leucine zippers, allow the quick isolation of a plurality of leucine zipper proteins.
  • structural tags (which may only be the proteins themselves) can allow heteromultimeric protein complexes to form, that then are assayed for activity as complexes. That is, many proteins, such as many eucaryotic transcription factors, function as heteromultimeric complexes which can be assayed using the present invention.
  • the candidate protein library may be a constructed library; that is, it may be generated using computational methods or built to contain only members of a defined class, or combinations of classes.
  • libraries of immunoglobulins may be built, or libraries of G-protein coupled receptors, tumor suppressor genes, proteases, transcription factors, phosphatases, kinases, etc.
  • a computational method is used to generate the candidate protein library.
  • the method is Protein Design Automation (PDA), as is described in U.S.S.N.s 60/061 ,097, 60/043,464, 60/054,678, 09/127,926, PCT US98/07254, 09/419,351 , and 09/927,790, all of which are expressly incorporated herein by reference.
  • PDA Protein Design Automation
  • a known protein structure is used as the starting point.
  • the residues to be optimized are then identified, which may be the entire sequence or subset(s) thereof.
  • the side chains of any positions to be varied are then removed.
  • the resulting structure consisting of the protein backbone and the remaining sidechains is called the template.
  • Each variable residue position is then preferably classified as a core residue, a surface residue, or a boundary residue; each classification defines a subset of possible amino acid residues for the position (for example, core residues generally will be selected from the set of hydrophobic residues, surface residues generally will be selected from the hydrophilic residues, and boundary residues may be either).
  • Each amino acid can be represented by a discrete set of all allowed conformers of each side chain, called rotamers.
  • all possible sequences of rotamers must be screened, where each backbone position can be occupied either by each amino acid in all its possible rotameric states, or a subset of amino acids, and thus a subset of rotamers.
  • Two sets of interactions are then calculated for each rotamer at every position: the interaction of the rotamer side chain with all or part of the backbone (the “singles” energy, also called the rotamer/template or rotamer/backbone energy), and the interaction of the rotamer side chain with all other possible rotamers at every other position or a subset of the other positions (the “doubles” energy, also called the rotamer/rotamer energy).
  • the energy of each of these interactions is calculated through the use of a variety of scoring functions, which include the energy of van der Waal's forces, the energy of hydrogen bonding, the energy of secondary structure propensity, the energy of surface area solvation and the electrostatics.
  • the total energy of each rotamer interaction, both with the backbone and other rotamers is calculated, and stored in a matrix form.
  • rotamer sets allow a simple calculation of the number of rotamer sequences to be tested.
  • a backbone of length n with m possible rotamers per position will have m n possible rotamer sequences, a number which grows exponentially with sequence length and renders the calculations either unwieldy or impossible in real time.
  • a "Dead End Elimination" (DEE) calculation is performed.
  • the DEE calculation is based on the fact that if the worst total interaction of a first rotamer is still better than the best total interaction of a second rotamer, then the second rotamer cannot be part of the global optimum solution.
  • a Monte Carlo search may be done to generate a rank- ordered or filtered list of sequences in the neighborhood of the DEE solution.
  • Starting at the DEE solution random positions are changed to other rotamers, and the new sequence energy is calculated. If the new sequence meets the criteria for acceptance, it is used as a starting point for another jump. After a predetermined number of jumps, a rank-ordered or filtered list of sequences is generated.
  • Monte Carlo searching is a sampling technique to explore sequence space around the global minimum or to find new local minima distant in sequence space. As is more additionally outlined below, there are other sampling techniques that can be used, including Boltzman sampling, genetic algorithm techniques and simulated annealing.
  • the kinds of jumps allowed can be altered (e.g. random jumps to random residues, biased jumps (to or away from wild- type, for example), jumps to biased residues (to or away from similar residues, for example), etc.).
  • the acceptance criteria of whether a sampling jump is accepted can be altered.
  • the protein backbone comprising (for a naturally occurring protein) the nitrogen, the carbonyl carbon, the ⁇ -carbon, and the carbonyl oxygen, along with the direction of the vector from the ⁇ -carbon to the ⁇ -carbon
  • the protein backbone structure contains at least one variable residue position.
  • the residues, or amino acids, of proteins are generally sequentially numbered starting with the N- terminus of the protein.
  • a protein having a methionine at it's N-terminus is said to have a methionine at residue or amino acid position 1 , with the next residues as 2, 3, 4, etc.
  • the wild type (i.e. naturally occurring) protein may have one of at least 20 amino acids, in any number of rotamers.
  • variant residue position herein is meant an amino acid position of the protein to be designed that is not fixed in the design method as a specific residue or rotamer, generally the wild-type residue or rotamer.
  • all of the residue positions of the protein are variable. That is, every amino acid side chain may be altered in the methods of the present invention. This is particularly desirable for smaller proteins, although the present methods allow the design of larger proteins as well. While there is no theoretical limit to the length of the protein which may be designed this way, there is a practical computational limit.
  • residue positions of the protein are variable, and the remainder are "fixed", that is, they are identified in the three dimensional structure as being in a set conformation.
  • a fixed position is left in its original conformation (which may or may not correlate to a specific rotamer of the rotamer library being used).
  • residues may be fixed as a non-wild type residue; for example, when known site-directed mutagenesis techniques have shown that a particular residue is desirable (for example, to eliminate a proteolytic site or alter the substrate specificity of an enzyme), the residue may be fixed as a particular amino acid.
  • the methods of the present invention may be used to evaluate mutations de novo, as js discussed below.
  • variable residues may be at least one, or anywhere from 0.1% to 99.9% of the total number of residues. Thus, for example, it may be possible to change only a few (or one) residues, or most of the residues, with all possibilities in between.
  • residues which can be fixed include, but are not limited to, structurally or biologically functional residues; alternatively, biologically functional residues may specifically not be fixed.
  • residues which are known to be important for biological activity such as the residues which form the active site of an enzyme, the substrate binding site of an enzyme, the binding site for a binding partner (ligand/receptor, antigen/antibody, etc.), phosphorylation or glycosylation sites which are crucial to biological function, or structurally important residues, such as disulfide bridges, metal binding sites, critical hydrogen bonding residues, residues critical for backbone conformation such as proline or glycine, residues critical for packing interactions, etc. may all be fixed in a conformation or as a single rotamer, or "floated".
  • residues which may be chosen as variable residues may be those that confer undesirable biological attributes, such as susceptibility to proteolytic degradation, dimerization or aggregation sites, glycosylation sites which may lead to immune responses, unwanted binding activity, unwanted allostery, undesirable enzyme activity but with a preservation of binding, etc.
  • each variable position is classified as either a core, surface or boundary residue position, although in some cases, as explained below, the variable position may be set to glycine to minimize backbone strain.
  • residues need not be classified, they can be chosen as variable and any set of amino acids may be used. Any combination of core, surface and boundary positions can be utilized: core, surface and boundary residues; core and surface residues; core and boundary residues, and surface and boundary residues, as well as core residues alone, surface residues alone, or boundary residues alone.
  • the classification of residue positions as core, surface or boundary may be done in several ways, as will be appreciated by those in the art.
  • the classification is done via a visual scan of the original protein backbone structure, including the side chains, and assigning a classification based on a subjective evaluation of one skilled in the art of protein modeling.
  • a preferred embodiment utilizes an assessment of the orientation of the C ⁇ -C ⁇ vectors relative to a solvent accessible surface computed using only the template C ⁇ atoms, as outlined in U.S.S.N.s 60/061 ,097, 60/043,464, 60/054,678, 09/127,926 and PCT US98/07254.
  • a surface area calculation can be done.
  • a set of amino acid side chains is assigned to each position. That is, the set of possible amino acid side chains that the program will allow to be considered at any particular position is chosen. Subsequently, once the possible amino acid side chains are chosen, the set of rotamers that will be evaluated at a particular position can be determined.
  • a core residue will generally be selected from the group of hydrophobic residues consisting of alanine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, and methionine (in some embodiments, when the ⁇ scaling factor of the van der Waals scoring function, described below, is low, methionine is removed from the set), and the rotamer set for each core position potentially includes rotamers for these eight amino acid side chains (all the rotamers if a backbone independent library is used, and subsets if a rotamer dependent backbone is used).
  • surface positions are generally selected from the group of hydrophilic residues consisting of alanine, serine, threonine, aspartic acid, asparagine, glutamine, glutamic acid, arginine, lysine and histidine.
  • the rotamer set for each surface position thus includes rotamers for these ten residues.
  • boundary positions are generally chosen from alanine, serine, threonine, aspartic acid, asparagine, glutamine, glutamic acid, arginine, lysine histidine, valine, isoleucine, leucine, phenylalanine, tyrosine, tryptophan, and methionine.
  • the rotamer set for each boundary position thus potentially includes every rotamer for these seventeen residues (assuming cysteine, glycine and proline are not used, although they can be). Additionally, in some preferred embodiments, a set of 18 naturally occurring amino acids (all except cysteine and proline, which are known to be particularly disruptive) are used.
  • proline, cysteine and glycine are not included in the list of possible amino acid side chains, and thus the rotamers for these side chains are not used.
  • the variable residue position has a ⁇ angle (that is, the dihedral angle defined by 1) the carbonyl carbon of the preceding amino acid; 2) the nitrogen atom of the current residue; 3) the ⁇ - carbon of the current residue; and 4) the carbonyl carbon of the current residue) greater than 0°
  • the position is set to glycine to minimize backbone strain.
  • This processing step entails analyzing interactions of the rotamers with each other and with the protein backbone to generate optimized protein sequences.
  • the processing initially comprises the use of a number of scoring functions to calculate energies of interactions of the rotamers, either to the backbone itself or other rotamers.
  • Preferred PDA scoring functions include, but are not limited to, a Van der Waals potential scoring function, a hydrogen bond potential scoring function, an atomic solvation scoring function, a secondary structure propensity scoring function and an electrostatic scoring function.
  • at least one scoring function is used to score each position, although the scoring functions may differ depending on the position classification or other considerations, like favorable interaction with an ⁇ -helix dipole.
  • the total energy which is used in the calculations is the sum of the energy of each scoring function used at a particular position, as is generally shown in Equation 1 :
  • Equation 1 the total energy is the sum of the energy of the van der Waals potential (E vdw ), the energy of atomic solvation (E as ), the energy of hydrogen bonding (E h . bond ⁇ ng ), the energy of secondary structure (E ss ) and the energy of electrostatic interaction (E etec ).
  • the term n is either 0 or 1, depending on whether the term is to be considered for the particular residue position.
  • the preferred first step in the computational analysis comprises the determination of the interaction of each possible rotamer with all or part of the remainder of the protein. That is, the energy of interaction, as measured by one or more of the scoring functions, of each possible rotamer at each variable residue position with either the backbone or other rotamers, is calculated. In a preferred embodiment, the interaction of each rotamer with the entire remainder of the protein, i.e.
  • portion refers to a fragment of that protein. This fragment may range in size from 10 amino acid residues to the entire amino acid sequence minus one amino acid.
  • portion refers to a fragment of that nucleic acid. This fragment may range in size from 10 nucleotides to the entire nucleic acid sequence minus one nucleotide.
  • the first step of the computational processing is done by calculating two sets of interactions for each rotamer at every position: the interaction of the rotamer side chain with the template or backbone (the “singles” energy), and the interaction of the rotamer side chain with all other possible rotamers at every other position (the “doubles” energy), whether that position is varied or floated.
  • the backbone in this case includes both the atoms of the protein structure backbone, as well as the atoms of any fixed residues, wherein the fixed residues are defined as a particular conformation of an amino acid.
  • “singles” (rotamer/template) energies are calculated for the interaction of every possible rotamer at every variable residue position with the backbone, using some or all of the scoring functions.
  • the hydrogen bonding scoring function every hydrogen bonding atom of the rotamer and every hydrogen bonding atom of the backbone is evaluated, and the E HB is calculated for each possible rotamer at every variable position.
  • the van der Waals scoring function every atom of the rotamer is compared to every atom of the template (generally excluding the backbone atoms of its own residue), and the E vdW is calculated for each possible rotamer at every variable residue position.
  • every atom of the first rotamer is compared to every atom of every possible second rotamer, and the E vdW is calculated for each possible rotamer pair at every two variable residue positions.
  • the surface of the first rotamer is measured against the surface of every possible second rotamer, and the E as for each possible rotamer pair at every two variable residue positions is calculated.
  • the secondary structure propensity scoring function need not be run as a "doubles" energy, as it is considered as a component of the "singles” energy. As will be appreciated by those in the art, many of these double energy terms will be close to zero, depending on the physical distance between the first rotamer and the second rotamer; that is, the farther apart the two moieties, the lower the energy.
  • feree fields that can be used in the PDA calculations can be used, including, but not limited to, Dreiding I and Dreiding II (Mayo et al, J. Phys. Chem. 948897 (1990)), AMBER (Weiner et al., J. Amer. Chem. Soc. 106:765 (1984) and Weiner et al., J. Comp. Chem. 106:230 (1986)), MM2 (Allinger J. Chem. Soc. 99:8127 (1977), Liljefors et al., J. Com. Chem. 8:1051 (1987)); MMP2 (Sprague et al., J.
  • cvff3.0 Disuber-Osguthorpe, et al.,(1988) Proteins: Structure, Function and Genetics, v4,pp31-47
  • cff91 Maple, et al., J. Comp. Chem. v15, 162-182
  • DISCOVER cvff and cff91
  • AMBER forcefields are used in the INSIGHT molecular modeling package (Biosym/MSI, San Diego California) and HARMM is used in the QUANTA molecular modeling package (Biosym/MSI, San Diego California), all of which are expressly incorporated by reference.
  • DEE Dead End Elimination
  • PDA viewed broadly, has three components that may be varied to alter the output (e.g. the primary library): the scoring functions used in the process; the filtering technique, and the sampling technique.
  • the scoring functions may be altered.
  • the scoring functions outlined above may be biased or weighted in a variety of ways. For example, a bias towards or away from a reference sequence or family of sequences can be done; for example, a bias towards wild-type or homologous residues may be used.
  • the entire protein or a fragment of it may be biased; for example, the active site may be biased towards wild-type residues, or domain residues towards a particular desired physical property can be done.
  • a bias towards or against increased energy can be generated.
  • Additional scoring function biases include, but are not limited to applying electrostatic potential gradients or hydrophobicity gradients, adding a substrate or binding partner to the calculation, or biasing towards a desired charge or hydrophobicity.
  • Additional scoring functions include, but are not limited to torsional potentials, or residue pair potentials, or residue entropy potentials.
  • additional scoring functions can be used alone, or as functions for processing the library after it is scored initially.
  • functions derived from data on binding of peptides to MHC can be used to rescore a library in order to eliminate proteins containing sequences which can potentially bind to MHC, i.e. potentially immunogenic sequences.
  • a variety of filtering techniques can be done, including, but not limited to, DEE and its related counterparts. Additional filtering techniques include, but are not limited to branch- and-bound techniques for finding optimal sequences (Gordon and Majo, Structure Fold. Des. 7:1089- 98, 1999), and exhaustive enumeration of sequences. It should be noted however, that some techniques may also be done without any filtering techniques; for example, sampling techniques can be used to find good sequences, in the absence of filtering.
  • sequence space sampling methods can be done, either in addition to the preferred Monte Carlo methods, or instead of a Monte Carlo search. That is, once a sequence or set of sequences is generated, preferred methods utilize sampling techniques to allow the generation of additional, related sequences for testing.
  • sampling methods can include the use of amino acid substitutions, insertions or deletions, or recombinations of one or more sequences.
  • a preferred embodiment utilizes a Monte Carlo search, which is a series of biased, systematic, or random jumps.
  • Monte Carlo search is a series of biased, systematic, or random jumps.
  • other sampling techniques including Boltzman sampling, genetic algorithm techniques and simulated annealing.
  • the kinds of jumps allowed can be altered (e.g. random jumps to random residues, biased jumps (to or away from wild- type, for example), jumps to biased residues (to or away from similar residues, for example), etc.).
  • the preferred methods of the invention result in a rank-ordered or filtered list of sequences; that is, the sequences are ranked or filtered on the basis of some objective criteria.
  • it is possible to create a set of non-ordered sequences for example by generating a probability table directly (for example using SCMF analysis or sequence alignment techniques) that lists sequences without ranking or filtering them.
  • the sampling techniques outlined herein can be used in either situation.
  • Boltzman sampling is done.
  • the temperature criteria for Boltzman sampling can be altered to allow broad searches at high temperature and narrow searches close to local optima at low temperatures (see e.g., Metropolis et al., J. Chem. Phys. 21 :1087, 1953).
  • the sampling technique utilizes genetic algorithms, e.g., such as those described by Holland (Adaptation in Natural and Artificial Systems, 1975, Ann Arbor, U. Michigan Press). Genetic algorithm analysis generally takes generated sequences and recombines them computationally, similar to a nucleic acid recombination event, in a manner similar to "gene shuffling". Thus the "jumps" of genetic algorithm analysis generally are multiple position jumps. In addition, as outlined below, correlated multiple jumps may also be done. Such jumps can occur with different crossover positions and more than one recombination at a time, and can involve recombination of two or more sequences. Furthermore, deletions or insertions (random or biased) can be done. In addition, as outlined below, genetic algorithm analysis may also be used after the secondary library has been generated.
  • Genetic algorithm analysis may also be used after the secondary library has been generated.
  • the sampling technique utilizes simulated annealing, e.g., such as described by Kirkpatrick et al. (Science, 220:671-680, 1983). Simulated annealing alters the cutoff for accepting good or bad jumps by altering the temperature. That is, the stringency of the cutoff is altered by altering the temperature. This allows broad searches at high temperature to new areas of sequence space, altering with narrow searches at low temperature to explore regions in detail.
  • libraries may also be subsequently mutated using known techniques (exposure to mutagens, error-prone PCR, error-prone transcription, combinatorial splicing (e.g. cre-lox recombination).
  • libraries of procaryotic and eukaryotic proteins may be made for screening in the systems described herein.
  • Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
  • SPA sequence prediction algorithm
  • the fusion nucleic acid can comprise the NAM enzyme and candidate protein in a variety of configurations, including both direct and indirect fusions, and include N- and C-terminal fusions and internal fusions.
  • the NAM enzyme and the candidate protein are directly fused.
  • a direct, in-frame fusion of the nucleic acid encoding the NAM enzyme and the candidate protein is engineered.
  • the library of fusion peptides can be constructed as N- and/or C-terminal fusions and internal fusions.
  • the NAM enzyme coding region may be 3' or 5' to the candidate protein coding region, or the candidate protein coding region may be inserted into a suitable position within the coding region of the NAM enzyme.
  • GFP green fluorescent protein
  • the NAM enzyme and the candidate protein are indirectly fused. This may be accomplished such that the components of the fusion remain attached, such as through the use of linkers, in ways that result in the components of the fusion becoming separated after translation, or, alternatively, in ways that start with the NAM enzyme and the candidate protein being made separately and then joined.
  • linkers may be used to functionally isolate the NAM enzyme and the candidate protein. That is, a direct fusion system may sterically or functionally hinder the interaction of the candidate protein with its intended binding partner, and thus fusion configurations that allow greater degrees of freedom are useful. An analogy is seen in the single chain antibody area, where the incorporation of a linker allows functionality. As will be appreciated by those in the art, there are a wide variety of different types of linkers that may be used, including cleavable and non-cleavable linkers; this cleavage may also occur at the level of the nucleic acid, or at the protein level.
  • linkers known to confer flexibility are used.
  • useful linkers include glycine-serine polymers (including, for example, (GS) n , and (GGGS) n (SEQ ID NO:51), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers such as the tether for the shaker potassium channel, and a large variety of other flexible linkers, as will be appreciated by those in the art.
  • Glycine-serine polymers are preferred since both of these amino acids are relatively unstructured, and therefore may be able to serve as a neutral tether between components.
  • serine is hydrophilic and therefore able to solubilize what could be a globular glycine chain.
  • similar chains have been shown to be effective in joining subunits of recombinant proteins such as single chain antibodies.
  • the linker used to construct indirect fusion enzymes can be a cleavable linker.
  • Cleavable linkers can function at the level of the nucleic acid or the protein. That is, cleavage (which in this sense means that the NAM enzyme and the candidate protein are separated) can occur during transcription, or before or after translation.
  • the cleavage can occur as a result of a cleavage functionality built into the nucleic acid.
  • cleavable nucleic acid sequences or sequences that will disrupt the nucleic acid, can be used.
  • the linkers are heterodimerization domains.
  • both the NAM enzyme and the candidate protein are fused to heterodimerization domains (or multimeric domains, if multivalency is desired), to allow association of these two proteins after translation.
  • cleavable protein linkers are used.
  • the fusion nucleic acids include coding sequences for a protein sequence that may be subsequently cleaved, generally by a protease.
  • protease directed to ubiquitous proteases, e.g. those that are constitutively present in most or all of the host cells of the system, can be used.
  • cleavage sites that correspond to cell-specific proteases may be used.
  • cleavage sites for proteases that are induced only during certain cell cycles or phases or are signal specific events may be used as well.
  • cleavable linkers include, but are not limited to, the prosequence of bovine chymosin, the prosequence of subtilisin, the 2a site (Ryan et al., J. Gen. Virol. 72:2727 (1991); Ryan et al., EMBO J.
  • Particular amino acid sites that serve as chemical cleavage sites include, but are not limited to, methionine for cleavage by cyanogen bromide (Shen, PNAS USA 81 :4627 (1984); Kempe et al., Gene 39:239 (1985); Kuliopulos et al., J. Am. Chem. Soc. 116:4599 (1994); Moks et al., Bio Technology 5:379 (1987); Ray et al., Bio/Technology 11 :64 (1993)), acid cleavage of an Asp-Pro bond (Wingender et al., J. Biol. Chem. 264(8):4367 (1989); Gram et al., Bio/Technology 12:1017 (1994)), and hydroxylamine cleavage at an Asn-Gly bond (Moks, supra).
  • the NAM enzyme and the candidate protein can be made separately and then joined later.
  • the nucleic acids encoding these components can be made separately and joined later as well.
  • the nucleic acids of the present invention can be expressed as c/s-fusions and as transfusions.
  • the expressed protein contains both the NAM enzyme (e.g. the Rep protein) and the candidate protein.
  • a fusion polypeptide is formed via transcription of a single messenger RNA.
  • the nucleic acids of the present invention also can be expressed as frar/s-fusions.
  • the NAM enzyme and the candidate protein are expressed separately as fusions with one or more merger moieties that allow later fusion; for example, a merger moiety can have the ability to participate in a ligation reaction, or have the ability to participate in a cross-linking reaction.
  • the resulting fusions are then joined to form a fusion protein in which the NAM enzyme is generally (but not required to be) covalently linked to the candidate protein.
  • Suitable ligation reactions include, but are not limited to, an intein catalyzed trans-ligation reaction.
  • a suitable cross-linking reaction is the cross-linking reaction catalyzed by transglutaminase.
  • the ligation reaction is an intein catalyzed trans-ligation reaction.
  • Inteins are self-splicing proteins that occur as in-frame insertions in specific host proteins.
  • inteins excise themselves from a precursor protein, while the flanking regions, the exteins, become joined via a new peptide bond to form a linear protein.
  • inteins are bifunctional proteins mediating both protein splicing and DNA cleavage.
  • Such elements consist of a protein splicing domain interrupted by an endonuclease domain. Because endonuclease activity is not required for protein splicing, mini-inteins, with accurate splicing activity can be generated by deletion of this central domain (Wood, et al., (1999) Nature Biotechnology, 17:889-892).
  • Protein splicing involves four nucleophilic displacements by three conserved splice junction residues. These residues, located near the intein/extein junctions, include the initial cysteine, serine, or threonine of the intein, which intiates splicing with an acyl shift.
  • Inteins also catalyze a trans-ligation reaction.
  • the ability of intein function to be reconstituted in trans by spatially separated intein domains suggests that the self-splicing motifs or mini inteins can be used to link any two peptides or polypeptides that are fused to the mini-inteins (Mills, et al., (1998) Proc. Natl. Acad. Sci, USA, 95:3543-3548).
  • inteins or “mini-inteins” or “intein motifs”, or “intein domains”, or grammatical equivalents herein is meant a protein sequence which, during protein splicing, is excised from a protein precursor.
  • the NAM enzyme fusion nucleic acid is designed with the primary sequence from the N-terminus of a suitable intein; thus the fusion nucleic acid comprise l N -NAM enzyme.
  • I N is defined herein as the N-terminal intein motif and the NAM enzyme is defined as described herein.
  • the candidate protein fusion nucleic acid is designed with the primary sequence from the C-terminus of a suitable intein; thus the fusion nucleic acid comprises l c -candidate protein.
  • I c is defined herein as the C-terminal intein motif and the candidate protein is defined as described above.
  • DNA sequences encoding the inteins may be obtained from a prokaryotic DNA sequence, such as a bacterial DNA sequence, or a eukaryotic DNA sequence, such as a yeast DNA sequence.
  • the Intein Registry includes a list of all experimental and theoretical inteins discovered to date and submitted to the registry (http://www.neb.com/inteins/int req.html).
  • fusion polypeptides are designed using intein motifs selected from organisms belonging to the Eucarya and Eubacteria, with the intein Ssp DnaB (GenBank accession number Q55418) being particularly preferred.
  • GenBank accession numbers for other intein proteins and nucleic acids include, but are not limited to: Ceu ClpP (GenBank acession number P42379); CIV RIR1 (T03053); Ctr VMA (GenBank accession number A46080); Gth DnaB (GenBank accession number 078411 ); Ppu DnaB (GenBank accession number P51333); See VMA (GenBank accession number PXBYVA); Mf1 RecA (GenBank accession number not given); Mxe GyrA (GenBank accession number P72065); Ssp DnaE (GenBank accession number S76958 & S75328); and Mle DnaB (GenBank accession number CAA17948.1)
  • inteins with alternative splicing mechanisms are preferred (see Southworth, et al., (2000) EMBO J., 19:5019-26).
  • GenBank accession numbers for inteins with alternative splicing mechanisms include, but are not limited to: Mja KlbA (GenBank accession number Q58191); and, Pfu KlbA (PF_949263 in UMBI).
  • thermophiles i.e., Archaea
  • Mth RIR1 GenBank accession number G69186
  • Pfu RIR1-1 AAB36947.1
  • Psp-GBD Pol GeneBank accession number AAA67132.1
  • Thy Pol-2 GeneBank accession number CAC18555.1
  • Pfu IF2 PF_1088001 in UMBI
  • Mja r-Gyr GenBank accession number G64488)
  • Pho RFC GeneBank accession number F71231)
  • Pab RFC-2 GeneBank accession number C75198
  • Mja RtcB also referred to as Mja Hyp-2; GenBank accession
  • transglutaminases catalyze protein-to-protein cross-linking reactions (Lorand. (1996) Proc. Natl. Acad. Sci. USA, 93:24310-14313).
  • the geometry of the cross-linked protein products depend that results from the cross-linking reaction depends on the number and spatial distribution of transglutaminase reactive glutamine and lysine residues in the protein substrates. Proteins with transglutaminase reactive glutamines are referred to as acceptor protein substrates, while proteins with lysine residues are referred to as donor protein substrates.
  • glutamine residues must be part of a peptide or polypeptide (Kahlem, P., et al., (1996) Proc. Natl. Acad. Sci. USA, 93:14580-14585). It has long been known that in certain small proteins, most or all scattered glutamine residues may act as amine acceptors, at least in the absence of secondary or tertiary structure preventing access of the enzyme. However, in native proteins, the nature of the neighboring residues has appreciable influence on the reactivity of a glutamine residue, with some residues being preferred to others. Among preferred glutamine residues are ones adjacent to as second glutamine residue.
  • a NAM enzyme-candidate protein fusion is made using a transglutaminase catalyzed cross-linking reaction.
  • polyglutamine residues may be added to the N- or C- terminus of either the NAM enzyme or the candidate protein to create an acceptor protein substrate. Between 1 and 6 glutamine residues may be added, with 2 residues being particularly preferred (Kahlem et al., supra).
  • Donor protein substrates can be created by adding a lysine residue to the N- or C- terminus of either the NAM enzyme or the candidate protein.
  • an acceptor donor substrate comprising a NAM enzyme with polyglutamine residues is combined with a donor substrate comprising a candidate protein with a lysine residue.
  • Cross-linking of the NAM enzyme to the candidate protein to form a fusion polypeptide is done under conditions that favor transglutaminase cross-linking (Kahlem et al., supra).
  • the cross-linking reaction may be carried out in vitro by adding purified transglutaminase or in vivo.
  • the EAS can be introduced into the nucleic acid molecule as two non-functional halves that are brought together following enzyme-mediated or non-enzyme-mediated homologous recombination, such as that mediated by cre-lox recombination, to form a functional EAS.
  • the referenced cre-lox consideration could also be used to control the formation of a functional fusion enzyme.
  • the control of cre-lox recombination is preferably mediated by introducing the recombinase gene under the control of an inducible promoter into the expression system, whether on the same nucleic acid molecule or on another expression vector.
  • the expression vectors can also include components to ease in the enrichment and identification process of "hits" identified using the methods of the invention, as is more fully described below.
  • the covalent linkage between the NAM enzyme and the EAS sequence of the vector hinders the enrichment process (generally done through PCR) after a candidate protein has been identified as a hit. Accordingly, this embodiment relies on the use of recombinases and recombinase sites such as the cre/lox system and the FLP system (see for example the CreatorTM Gene Cloning and Expression System sold by Clontech and the GatewayTM cloning system from Life Technologies, Inc.
  • the recombinase sites e.g. the lox sites
  • the recombinase sites are inserted downstream of the fusions (either prior to the creation of the fusions or afterwards).
  • Panning and/or assays are run, as generally described below, to identify "hits".
  • These positive clone pools are purified (for example through phenol extraction and ethanol precipitation) and mixed with fresh vectors in the presence of the corresponding recombinase (for example the cre recombinase when lox sites are used).
  • the fusion nucleic acids can comprise additional coding sequences for other functionalities.
  • the discussion herein is directed to fusions of these other components to the fusion nucleic acids described herein; however, they can also be separate from the fusion protein and rather be a component of the expression vector comprising the fusion nucleic acid, as is generally outlined below.
  • the fusions are linked to a fusion partner.
  • fusion partner or “functional group” herein is meant a sequence that is associated with the candidate protein that confers upon all members of the library in that class a common function or ability. Fusion partners can be heterologous (i.e. not native to the host cell), or synthetic (not native to any cell).
  • Suitable fusion partners include, but are not limited to: a) presentation structures, as defined below, which provide the candidate proteins in a conformationally restricted or stable form, including hetero- or homodimerization or multimerization sequences; b) rescue sequences as defined below, which allow the purification or isolation of the NAP conjugates; c) stability sequences, which confer stability or protection from degradation to the candidate protein or the nucleic acid encoding it, for example resistance to proteolytic degradation; d) linker sequences; or e) any combination of a), b), c), and d), as well as linker sequences as needed.
  • the fusion partner is a presentation structure.
  • presentation structure or grammatical equivalents herein is meant an amino acid sequence, which, when fused to candidate proteins, causes the candidate proteins to assume a conformationally restricted form. This is particularly useful when the candidate proteins are random, biased random or pseudorandom peptides. Proteins interact with each other largely through conformationally constrained domains. Although small peptides with freely rotating amino and carboxyl termini can have potent functions as is known in the art, the conversion of such peptide structures into pharmacologic agents is difficult due to the inability to predict side-chain positions for peptidomimetic synthesis.
  • synthetic presentation structures i.e. artificial polypeptides
  • synthetic presentation structures are capable of presenting a randomized peptide as a conformationally-restricted domain.
  • presentation structures comprise a first portion joined to the N-terminal end of the randomized peptide, and a second portion joined to the C-terminal end of the peptide; that is, the peptide is inserted into the presentation structure, although variations may be made, as outlined below.
  • the presentation structures are selected or designed to have minimal biologically activity when expressed in the target cell.
  • suitable presentation structures maximize accessibility to the peptide by presenting it on an exterior loop.
  • suitable presentation structures include, but are not limited to, minibody structures, dimerization sequences, loops on beta-sheet turns and coiled-coil stem structures in which residues not critical to structure are randomized, zinc-finger domains, cysteine-linked (disulfide) structures, transglutaminase linked structures, cyclic peptides, B-loop structures, helical barrels or bundles, leucine zipper motifs, etc.
  • the presentation structure is a coiled-coil structure, allowing the presentation of the randomized peptide on an exterior loop. See, for example, Myszka et al., Biochem. 33:2362-2373 (1994), hereby incorporated by reference. Using this system investigators have isolated peptides capable of high affinity interaction with the appropriate target. In general, coiled-coil structures allow for between 6 to 20 randomized positions. A preferred coiled-coil presentation structure is described in, for example, Martin et al., EMBO J. 13(22):5303-5309 (1994), incorporated by reference.
  • the presentation structure is a minibody structure.
  • a "minibody” is essentially composed of a minimal antibody complementarity region.
  • a preferred minibody presentation structure is as follows:
  • MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRYIVSRDTSQSILYLQKKKG PP (SEQ ID NO:52).
  • the bold, underlined regions are the regions which may be randomized.
  • the italicized phenylalanine must be invariant in the first randomizing region.
  • the entire peptide is cloned in a three-oligonucleotide variation of the coiled-coil embodiment, thus allowing two different randomizing regions to be incorporated simultaneously.
  • This embodiment utilizes non-palindromic BstXI sites on the termini.
  • the presentation structure is a sequence that contains generally two cysteine residues, such that a disulfide bond may be formed, resulting in a conformationally constrained sequence.
  • This embodiment is particularly preferred when secretory targeting sequences are used.
  • any number of random sequences, with or without spacer or linking sequences, may be flanked with cysteine residues.
  • effective presentation structures may be generated by the random regions themselves.
  • the random regions may be "doped" with cysteine residues which, under the appropriate redox conditions, may result in highly crosslinked structured conformations, similar to a presentation structure.
  • the randomization regions may be controlled to contain a certain number of residues to confer ⁇ -sheet or a-helical structures.
  • the presentation structure is a dimerization or multimerization sequence.
  • a dimerization sequence allows the non-covalent association of one candidate protein to another candidate protein, including peptides, with sufficient affinity to remain associated under normal physiological conditions. This effectively allows small libraries of candidate protein (for example, 10 4 ) to become large libraries if two proteins per cell are generated which then dimehze, to form an effective library of 10 8 (10 4 X 10 4 ). It also allows the formation of longer proteins, if needed, or more structurally complex molecules.
  • the dimers may be homo- or heterodimers.
  • Dimerization sequences may be a single sequence that self-aggregates, or two sequences. That is, nucleic acids encoding both a first candidate protein with dimerization sequence 1 , and a second candidate protein with dimerization sequence 2, such that upon introduction into a cell and expression of the nucleic acid, dimerization sequence 1 associates with dimerization sequence 2 to form a new structure.
  • dimerization sequences will encompass a wide variety of sequences. Any number of protein- protein interaction sites are known. In addition, dimerization sequences may also be elucidated using standard methods such as the yeast two hybrid system, traditional biochemical affinity binding studies, or even using the present methods.
  • the fusion partner is a rescue sequence (sometimes also referred to herein as "purification tags” or "retrieval properties").
  • a rescue sequence is a sequence which may be used to purify or isolate either the candidate protein or the NAP conjugate.
  • peptide rescue sequences include purification sequences such as the His 6 tag for use with Ni affinity columns and epitope tags for detection, immunoprecipitation or FACS (fluoroscence-activated cell sorting).
  • Suitable epitope tags include myc (for use with the commercially available 9E10 antibody), the BSP biotinylation target sequence of the bacterial enzyme BirA, flu tags, lacZ, and GST.
  • Rescue sequences can be utilized on the basis of a binding event, an enzymatic event, a physical property or a chemical property.
  • the fusion partner is a stability sequence to confer stability to the candidate protein or the nucleic acid encoding it.
  • peptides can be stabilized by the incorporation of glycines after the initiation methionine, for protection of the peptide to ubiquitination as per Varshavsky's N-End Rule, thus conferring long half-life in the cytoplasm.
  • two pralines at the C-terminus impart peptides that are largely resistant to carboxypeptidase action. The presence of two glycines prior to the pralines impart both flexibility and prevent structure initiating events in the di- proline to be propagated into the candidate protein structure.
  • preferred stability sequences are as follows: MG(X) n GGPP (SEQ ID NO:53), where X is any amino acid and n is an integer of at least four.
  • linker sequences as defined above, may be used in any configuration as needed.
  • the fusion partners including presentation structures, may be modified, randomized, and/or matured to alter the presentation orientation of the randomized expression product.
  • determinants at the base of the loop may be modified to slightly modify the internal loop peptide tertiary structure, which maintaining the randomized amino acid sequence.
  • Combinations of fusion partners can be used if desired.
  • any number of combinations of presentation structures, rescue sequences, and stability sequences may be used, with or without linker sequences.
  • the fusion partners may be associated with any component of the expression vectors described herein: they may be directly fused with either the NAM enzyme, the candidate protein, or the EAS, described below, or be separate from these components and contained within the expression vector.
  • the nucleic acids of the invention preferably comprise an enzyme attachment sequence.
  • enzyme attachment sequence or “EAS” herein is meant selected nucleic acid sequences that mediate attachment with NAM enzymes.
  • EAS nucleic acid sequences possess the specific sequence or specific chemical or structural configuration that allows for attachment of the NAM enzyme and the EAS.
  • the EAS can comprise DNA or RNA sequences in their natural conformation, or hybrids.
  • EASs also can comprise modified nucleic acid sequences or synthetic sequences inserted into the nucleic acid molecule of the present invention.
  • EASs also can comprise non-natural bases or hybrid non-natural and natural (i.e., found in nature) bases.
  • EAS EAS
  • NAM/EAS pairs are the sequences recognized by Rep proteins (sometimes referred to herein as "Rep EASs") and the Rep proteins, the H-1 recognition sequence and H-1, etc.
  • Rep proteins sometimes referred to herein as "Rep EASs”
  • Rep proteins the Rep proteins
  • H-1 recognition sequence the H-1 recognition sequence
  • H-1 the H-1 recognition sequence
  • EASs can be utilized which mediate improved covalent linkage with the NAM enzyme compared to the wild-type or naturally occurring EAS.
  • the EAS is double-stranded.
  • a suitable EAS is a double-stranded nucleic acid sequence containing specific features for interacting with corresponding NAM enzymes.
  • Rep68 and Rep78 recognize an EAS contained within an AAV ITR, the sequence of which is set forth in Figure 50A (SEQ ID NO:57).
  • these Rep proteins have been shown to recognize an ITR-like region in human chromosome 19 as well, the sequence of which is shown in Figure 48 (SEQ ID NO:48).
  • An EAS also can comprise supercoiled DNA with which a topoisomerase interacts and forms covalent intermediate complexes.
  • an EAS is a restriction enzyme site recognized by an altered restriction enzyme capable of forming covalent linkages.
  • an EAS can comprise an RNA sequence and/or structure with which specific proteins interact and form stable complexes (see, for example, Romaniuk and Uhlenbeck, Biochemistry, 24, 4239-44 (1985)).
  • the EAS is an RNA sequence and RNA-protein fusions are made.
  • RNA-protein fusions are made by fusing a gene encoding a NAM enzyme (described above) to either the N- or C-terminal of a gene encoding a candidate protein to create a fusion nucleic acid.
  • An EAS specific for the NAM enzyme may be inserted in either the 5' UTR and/or the 3' UTR of the fusion nucleic acid. As shown in Figure 51, as the fusion nucleic acid is translated, the newly translated NAM protein covalently binds to the EAS, thereby creating an RNA-protein fusion.
  • the present invention relies on the specific binding of the NAM enzyme to the EAS in order to mediate linkage of the fusion enzyme to the nucleic acid molecule.
  • the EAS of the present invention is preferably comprised of a nucleic acid sequence of sufficient length such that specific fusion protein-coding nucleic acid molecule attachment results.
  • the EAS is preferably greater than five nucleotides in length. More preferably, the EAS is greater than 10 nucleotides in length, e.g., with EASs of at least 12, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides being preferred.
  • the EAS comprises at least 165 base pairs and has the sequence set forth in Figure 50B (SEQ ID NO:58).
  • the EAS comprises at least 80 base pairs and has the sequence set forth in Figure 50C (SEQ ID NO:59).
  • the EAS comprises at least 50 base pairs and has the sequence set forth in Figure 50D (SEQ ID NO:60).
  • the NAM enzyme used in the present invention or the corresponding EAS can be manipulated in order to increase the stability of the fusion protein- nucleic acid molecule complex. Such manipulations are contemplated herein, so long as the NAM enzyme forms a covalent bond with its corresponding EAS.
  • the nucleic acids of the invention preferably comprise a DNA binding motif.
  • DNA binding motif herein is meant selected nucleic acid sequences that mediate attachment of small molecule conjugates.
  • the DNA binding motif should posses a sequence, or a specific chemical or structural configuration to allow for the attachment of a small molecule conjugate.
  • the DNA binding motif may comprise DNA sequences in their natural conformation or hybrids.
  • the DNA binding motif also can comprise modified nucleic acid sequences or synthetic sequences, non-natural bases or hybrid non-natural and natural bases.
  • DNA binding motifs include, but are not limited to, binding sequences capable of binding small molecule conjugates; for example, molecules that can be combined in antiparallel, side-by-side, dimeric complexes or in hairpin or cyclic configurations.
  • DNA binding motifs are between 4 to 20 base pairs.
  • the DNA binding motifs of the present invention may be one of any of the following lengths: 4 base pairs, 5 base pairs, 6 base pairs, 7 base pairs, 8 base pairs, 9 base pairs, 10 base pairs, 11 base pairs, 12 base pairs, 13 base pairs, 14 base pairs, 15 base pairs, 16 base pairs, 17 base pairs, 18 base pairs, 19 base pairs, and 20 base pairs in length.
  • Binding motifs of 5 to 7 base pairs are advantageous as binding affinity for small molecule conjugates, especially polyamides, is high. See Dervan and B ⁇ rli, (1999) Curr. Opin. Chem. Biol. 3:688-693, hereby incorporated by reference in its entirety.
  • the DNA sequence of the binding motif comprises (A/T)G(AtT)C(A/T).
  • Other suitable DNA sequences include, but are not limited to, (A/T)G(AT) 3 ; GTACA; TGTACA; TGTGTA; TGTAACA; TGTTATTGTTA (SEQ ID NO:54); and other suitable sequences described in Dervan and B ⁇ rli, supra; Mapp, et al., (2000) Proc. Natl. Acad. Sci. USA, 97:3930-3935.
  • small molecule conjugate herein is meant a small molecule that comprises at least two domains.
  • the first domain comprises a moiety capable of recognizing DNA in a sequence specific manner, referred to herein as a "DNA binding moiety".
  • DNA binding moiety is synthetic ligand that recognizes and binds too DNA. That is, the ligand is capable of recognizing and binding to specific sequences in either the major or minor groove of DNA (Dervan and B ⁇ rli, supra).
  • the synthetic ligand will recognize and bind to the minor groove of DNA.
  • Suitable ligands for binding to the minor groove of DNA include, but are not limited to polyamides.
  • Suitable polyamides include, but are not limited to, synthetic peptides containing non-natural amino acids, N-tmethyl-imidazole, N-methyl-pyrrole, N-methyl-3-hydroxypyrrole (Hp), and the amino acid beta-alanine.
  • Synthetic ligands are preferably designed using the pairing rules for polyamide binding to DNA (Dervan and B ⁇ rli, supra.)
  • a pyrrole (Py) opposite an imidazole (Im; Py/lm pairing) targets a C-G base pair (bp)
  • an Im/Py pair recognizes a G- C bp/ A Py/Py pair is degenerated and binds both A-T and T-A pairs in preference to G-C/C-G pairs.
  • the A-T/T-A degeneracy by Py/Py can be avoided by using an Hp/Py pair.
  • An Hp/Py pair recognizes a T-A bp whereas a Py/Hp pair targets an A-T bp.
  • Synthetic ligands comprising polyamides may be synthesized as cyclic or hairpin structures, tandem hairpins, H-pins, or as unlinked dimers (homo or heterodimers). Hairpin structures are preferred, as they provide high affinity and specificity, especially as the number of heterocyclic units are increased. Hairpin structures may be created by connecting the carboxyl and amino terminal of two adjacent polyamides with a ⁇ -butyric acid linker (see disclosure 2 paragraphs below and conform e.g. chiral). A carboxy-terminal ⁇ -linker element, such as a ⁇ -alanine reside may be used to specify for A-T in preference to G-C (Dervan and B ⁇ rli, supra) with increased DNA affinity.
  • hairpin structures of core sequence composition lmPyPy- ⁇ -PyPy may be used coding to G A/T A/T A T.
  • Other useful hairpin structures have core sequence compositions comprising eight Im and Py rings linked with a ⁇ -butyric acid linker and terminate in a ⁇ -alanine residue.
  • hairpin structures may be created using Hp-lm-Py motifs.
  • cooperatively binding hairpin polyamide ligands which bind in a homo or hetero dimeric fashion can be designed (see Dervan and B ⁇ rli, supra).
  • synthetic ligands containing Im and Py are combined in anti-parallel, unlinked side-by-side dimeric complexes, which may consist of homo or hetero dimers, for the recognition of longer sequences.
  • a ⁇ -alanine residue can be used to join adjacent polyamide subunits to provide fully overlapping or partially overlapping extended homodimers recognizing between 10 to 20 bp (see Dervan and B ⁇ rli, supra).
  • chiral turn, cyclic or ⁇ /ring pair polyamide synthetic ligands can be designed. These ligands are especially used for binding to DNA sequences that exhibit microstructure (see Dervan and B ⁇ rli, supra).
  • the second domain comprises a "rescue tag" as defined below.
  • the two domains may be contiguous or separated by linker sequence as defined below.
  • rescue sequences can rely on the use of triplex helix formation, with high stabilities, using naturally occurring nucleosides of analogs such as PNA.
  • the fusion nucleic acids can also comprise capture sequences that hybridize to capture probes on a surface, to allow the formation of support bound NAP conjugates and specifically arrays of the conjugates.
  • the expression vectors may comprise a number of additional components, including, selection genes as outlined herein (particularly including growth-promoting or growth-inhibiting functions), activatible elements, recombination signals (e.g. cre and lox sites) and labels.
  • the present invention fusion peptide, fusion nucleic acid, conjugates, etc. further comprise a labeling component.
  • the label can be fused to one or more of the other components, for example to the NAM fusion protein, in the case where the NAM enzyme and the candidate protein remain attached, or to either component, in the case where scission occurs, or separately, under its own promoter.
  • other components of the assay systems may be labeled.
  • Labels can be either direct or indirect detection labels, sometimes referred to herein as “primary” and “secondary” labels.
  • detection label or “detectable label” herein is meant a moiety that allows detection. This may be a primary label or a secondary label. Accordingly, detection labels may be primary labels (i.e. directly detectable) or secondary labels (indirectly detectable).
  • labels fall into four classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic, electrical, thermal labels; c) colored or luminescent dyes or moieties; and d) binding partners. Labels can also include enzymes (horseradish peroxidase, etc.) and magnetic particles.
  • the detection label is a primary label.
  • a primary label is one that can be directly detected, such as a fluorophore.
  • Preferred labels include, for example, chromophores or phosphors but are preferably fluorescent dyes or moieties.
  • Fluorophores can be either "small molecule" fluors, or proteinaceous fluors.
  • suitable dyes for use in the invention include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, quantum dots (also referred to as "nanocrystals"), pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, Cy dyes (Cy3, Cy5, etc.), alexa dyes, phycoerythin, bodipy, and others described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland, hereby expressly
  • proteinaceous fluores are used.
  • Suitable autofluorescent proteins include, but are not limited to, the green fluorescent protein (GFP) from Aequorea and variants thereof; including, but not limited to, GFP, (Chalfie, et al., Science 263(5148):802-805 (1994)); enhanced GFP (EGFP; Clontech - Genbank Accession Number U55762 )), blue fluorescent protein (BFP; Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal (Quebec) Canada H3H 1J9; Stauber, R. H.
  • GFP green fluorescent protein
  • the label protein is Aequorea green fluorescent protein or one of its variants; see Cody et al., Biochemistry 32:1212-1218 (1993); and Inouye and Tsuji, FEBS Lett. 341 :277-280 (1994), both of which are expressly incorporated by reference herein.
  • a secondary detectable label is used.
  • a secondary label is one that is indirectly detected; for example, a secondary label can bind or react with a primary label for detection, can act on an additional product to generate a primary label (e.g. enzymes), or may allow the separation of the compound comprising the secondary label from unlabeled materials, etc.
  • Secondary labels include, but are not limited to, one of a binding partner pair; chemically modifiable moieties; enzymes such as horseradish peroxidase, alkaline phosphatases, luciferases, etc; and cell surface markers, etc.
  • the secondary label is a binding partner pair.
  • the label may be a hapten or antigen, which will bind its binding partner.
  • the binding partner can be attached to a solid support to allow separation of components containing the label and those that do not.
  • suitable binding partner pairs include, but are not limited to: antigens (such as proteins (including peptides)) and antibodies (including fragments thereof (FAbs, etc.)); proteins and small molecules, including biotin/streptavidin; maltose binding protein/amylose beads; enzymes and substrates or inhibitors; other protein-protein interacting pairs; receptor-ligands; and carbohydrates and their binding partners.
  • Nucleic acid - nucleic acid binding proteins pairs are also useful. In general, the smaller of the pair is attached to the system component for incorporation into the assay, although this is not required in all embodiments.
  • Preferred binding partner pairs include, but are not limited to, biotin (or imino-biotin) and streptavidin, digeoxinin and Abs, etc.
  • the binding partner pair comprises a primary detection label (for example, attached to the assay component) and an antibody that will specifically bind to the primary detection label.
  • specifically bind herein is meant that the partners bind with specificity sufficient to differentiate between the pair and other components or contaminants of the system. The binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding.
  • the dissociation constants of the pair will be less than about lO ' O "6 M " ⁇ with less than about 10 "5 -10 9 M ⁇ 1 , being preferred and less than about 10 "7 - 10 9 M 1 being particularly preferred.
  • the secondary label is a chemically modifiable moiety.
  • labels comprising reactive functional groups are incorporated into the assay component.
  • the functional group can then be subsequently labeled with a primary label.
  • Suitable functional groups include, but are not limited to, amino groups, carboxy groups, maleimide groups, oxo groups and thiol groups, with amino groups and thiol groups being particularly preferred.
  • primary labels containing amino groups can be attached to secondary labels comprising amino groups, for example using linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
  • the nucleic acids of the invention comprise (i) a fusion nucleic acid comprising sequences encoding a NAM enzyme and a candidate protein, and (ii) an EAS.
  • These nucleic acids are preferably incorporated into an expression vector; thus providing libraries of expression vectors, sometimes referred to herein as "NAM enzyme expression vectors”.
  • the expression vectors may be either self-replicating extrachromosomal vectors, vectors which integrate into a host genome, or linear nucleic acids that may or may not self-replicate.
  • linear nucleic acid molecules specifically included within the definition of expression vectors are linear nucleic acid molecules.
  • Expression vectors thus include plasmids, episomes, transposons and phage vectors.
  • the nucleic acid molecule and any of these expression vectors may be derived from natural or synthetic origins and be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for a chosen prokaryote host include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Sequence that are not directly related to expression in a given host may also be included, such as sequences derived from prokaryotic, eukaryotic and viral sources, alone or in combination.
  • sequences derived from prokaryotic, eukaryotic and viral sources may also be included, such as sequences derived from prokaryotic, eukaryotic and viral sources, alone or in combination.
  • the inclusion of such sequences may be designed for a variety of reasons, including but not limited to, allowing a given vector to be functional in prokaryotic and/or eukaryotic and/or viral systems.
  • An example of such a dual vector is pDualTM (Clontech) vector which allow for protein expression in both proKaryotic and eukaryotic organisms.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a presequence or secretory leader is operably linked to DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
  • a promoter is operably linked to a coding sequence if it affects the transcription of the sequence;
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
  • the transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the NAM protein, as will be appreciated by those in the art; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the NAM protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
  • the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and operator sequences.
  • the regulatory sequences include a promoter and transcriptional start and stop sequences.
  • a “promoter” is a nucleic acid sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis.
  • a suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of the fusion into mRNA.
  • a bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences.
  • Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose and maltose, and sequences derived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences. Furthermore, a bacterial promoter can include naturally occurring promoters of non- bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. The promoters can be either naturally occurring promoters, hybrid promoters, or synthetic promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
  • the expression vectors use a T7 promoter and at least one lac operator.
  • Alternative embodiments include the use of a T5 promoter and at least one lac operator; a TAC promoter and at least one lac operator; and a araBAD promoter (Invitrogen).
  • the ribosome binding site is called the Shine-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation codon.
  • SD sequences such as RBSII, may also be used.
  • the expression vector may also include a signal peptide sequence that provides for secretion of the fusion proteins in bacteria or other cells.
  • the signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art.
  • the protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
  • the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed.
  • Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline.
  • Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.
  • the expression vector comprises an origin of replication, such as ColE1 , pMB1 , p15A, pSC101, pPS10, P1 , RK2/RP4, R6K, R1 , ColE2-type (all of which are described in del Solar, G., et. al., (1998), Microbiology and Molecular Biology Review, 62:434-464, incorporated herein by reference in its entirety).
  • an origin of replication such as ColE1 , pMB1 , p15A, pSC101, pPS10, P1 , RK2/RP4, R6K, R1 , ColE2-type (all of which are described in del Solar, G., et. al., (1998), Microbiology and Molecular Biology Review, 62:434-464, incorporated herein by reference in its entirety).
  • Vectors suitable for both gram positive and gram negative organisms are of use in the methods of the invention.
  • Suitable vectors include, for example, vectors for use Bacillus subtilis, Escherichia coli, Streptococcus cremoris, Streptococcus lividans, Staphylococcus aureus, Streptococcus pneumoniae, Hemophilus influenzae, Moraxella catarrhalis and M. catt, among others.
  • the bacterial expression vectors can be transformed into commercially available bacterial host cells that have been made chemically or electrically competent (Invitrogen, Stratagene, or Novagen). Additionally, phage infection also can be used as a means of introducing the expression vectors into a bacterial host.
  • a library of procaryotic expression vectors comprising a fusion nucleic acid comprising a nucleic acid encoding a NAM enzyme and a candidate protein and an EAS is generated.
  • the procaryotic expression vector should be suitable for use in E. coli.
  • plasmid based or transposon based procaryotic expression vectors are used, although phage vectors may also be used.
  • the procaryotic expression vector is a plasmid expression vector.
  • plasmid expression vector herein is meant at a vector comprising a variety of components, including but not limited to, an origin of replication, a T7 promoter, a lac operator, a selectable marker gene and cloning sites for the introduction of a fusion nucleic acid comprising a NAM enzyme and a candidate protein and an EAS.
  • the vector also may comprise additional elements such as an f1 origin and fusion tags (such as His tags, etc.,) for the purification of the candidate protein.
  • Expression vectors with other promoters, such as the T5 promoter, araC promoter also may be used.
  • the plasmid expression vector is pET-24a(+) (Novagen) or a pET-24a(+)-like vector.
  • a fusion nucleic acid encoding a NAM enzyme and a candidate protein is introduced into the multiple cloning site of pET-24a(+) such that expression of the NAM/candidate protein is under the control of the T7 promoter.
  • the advantages of this approach are: 1) it allows the construction of the library in strains of E. coli such as Top 10, DH10B, DH5 ⁇ (Invitrogen), without background expression of the NAM/candidate protein fusion; and 2) it avoids the expression of genes from eucaryotes that may be toxic to procaryotes during the library construction stage.
  • the T7 promoter of pET-24a(+) is operably linked to a fusion nucleic acid comprising the Rep 68 variant, PCD302 fused to a nucleic acid encoding a candidate protein and an EAS inserted downstream from PCD302 (see Figure 54).
  • the EAS sequence may be inserted upstream, downstream, inside or outside of the transcriptional unit of PCD302.
  • the T7 promoter of a GatewayTM compatible vector Invitrogen; is operably linked to a fusion nucleic acid encoding a PCD302 and a candidate protein (See Figure 60A).
  • the EAS sequence may be inserted upstream, downstream, inside or outside of the transcriptional unit of PCD302.
  • the plasmid expression vector is based on pQE82L (Qiagen).
  • the T5 promoter is operably linked to a fusion nucleic acid encoding PCD302 and a candidate protein.
  • the EAS sequence may be inserted upstream, downstream, inside or outside of the transcriptional unit of PCD302 (see Figure 53).
  • the plasmid expression vector is based on pBAD//Wyc-His A, (Invitrogen).
  • the araBAD promoter is operably linked to a fusion nucleic acid encoding PCD302 and a candidate protein (see Figure 56).
  • the EAS sequence may be inserted upstream, downstream, inside or outside of the transcriptional unit of PCD302.
  • the procaryotic expression vector comprises a nucleic acid encoding a transposon.
  • transposon herein is meant nucleic acid sequence that is mobile and carries gene(s) that code for the enzyme activities required for transposition, i.e., transposases, although other genes may also be present.
  • transposable elements including insertion sequences and composite transposons, have been identified.
  • IS elements are autonomous units, each of which codes only for the protein needed to sponsor its own transposition.
  • an IS element ends in short inverted terminal repeats, that flank a single coding region for the transposase. See Benjamin Lewin, (2000) Genes VII, Chapter 15, Transposons, Oxford University Press, Oxford, pp.457-484.
  • Composite transposons in addition to having IS elements, carry drug resistance markers.
  • Composite transposons have two arms, that consist of the IS elements.
  • the IS elements code for transposase activities that are responsible for creating a target site and for recognizing the ends of the transposon. Only the ends are needed for a transposon to serve as a substrate for transposition.
  • the central region codes for transposon markers, such as genes that code for resistance to antibiotics.
  • any transposon capable of transposing in a procaryotic host may be used, including both IS elements and composite transposons.
  • composite transposons such as Tn5 or Tn10 are used.
  • the transposon may be modified in any number of ways to allow for the selection and recovery of NAM/candidate protein fusions. Such modifications may include the addition of nucleic acid sequences encoding selectable markers, EAS, a NAM enzyme operably linked to a promoter as described herein.
  • modifications to the IS elements are also made. For example, the IS elements may be removed entirely. Alternatively, only the outside and/or inside ends may be used (see Ehrmann, M., et al., (1997) Proc. Natl. Acad.
  • mini-transposon based on the Tn5 transposon are used (see Ehrmann, M., et al., (1997) Proc. Natl. Acad. Sci. USA, 94:13111-13115; Hensel, M., et al., (1995), Science, 269:400-403, incorporated herein by reference).
  • mini-transposon herein is meant a transposon comprising a portion of the IS element, such as the outside or inside end, a multiple cloning region, and a selectable marker.
  • Mini-transposons may be carried on any suitable plasmid expression vector, including suicide vectors (Hensel, M., et al., (1995), Science, 269:400-403) and plasmid vectors containing Tn5 transposase (Ehrmann, M., et al., (1997) Proc. Natl. Acad. Sci. USA, 94:13111-13115).
  • Suitable suicide vectors for use in gram negative bacteria, such as E. coli include pKNOCK (Alexeyev, MF, (1999) Biotechniques, 26:824-6, 828); pMAKSAC (Favre, D.
  • procaryotic expression vector constructs comprising transposon sequences are used.
  • a construct would include the following components: an origin of replication for R6K, a NAM enzyme under the control of a promoter, an EAS, an antibiotic marker, nucleic acid sequences encoding the inside and outside edge of Tn5 (see Figure 52). Additional components include a transposase gene.
  • nucleic acid sequences encoding IS elements from Tn10 may be used in place of the nucleic acid sequences encoding the inside and outside edge of Tn5.
  • the expression vectors of the invention can follow one of two fates, which are merely exemplary: they are introduced into cell-free translation systems, to create libraries of nucleic acid/protein (NAP) conjugates that are assayed in vitro, or, preferably they are introduced into host cells where the NAP conjugates are formed, the cells may be optionally lysed and assayed accordingly
  • NAP nucleic acid/protein
  • the expression vectors are made and introduced into cell-free systems for translation, followed by the attachment of the NAP enzyme to the EAS, forming a nucleic acid/protein (NAP) conjugate
  • NAP nucleic acid/protein conjugate
  • NAP conjugate a covalent attachment between the NAP enzyme and the EAS, such that the expression vector comprising the EAS is covalently attached to the NAP enzyme
  • Suitable cell free translation systems are known in the art
  • the expression vectors of the invention are introduced into host cells by electroporation
  • introduction into or grammatical equivalents herein is meant that the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid
  • the cells are optionally lysed Cell lysis is accomplished by any suitable technique, such as any of a variety of techniques known in the art (see, for example, Sambrook et al , Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N Y (1989), and Ausubel et al , Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N Y (1994), hereby expressly incorporated by reference) Most methods of cell lysis involve exposure to chemical, enzymatic, or mechanical stress Although the attachment of the fusion enzyme to its coding nucleic acid molecule is a covalent linkage, and can therefore withstand more varied conditions than non-covalent bonds, care should be taken to ensure that the fusion enzyme- nucleic acid molecule complexes remain intact, i e , the fusion enzyme remains associated with the expression vector
  • the NAP conjugate may be purified or isolated after lysis of the cells Ideally, the lysate containing the fusion protein-nucleic acid molecule complexes is separated from a majority of the resulting cellular debris in order to facilitate interaction with the target
  • the NAP conjugate may be isolated or purified away from some or all of the proteins and compounds with which it is normally found after expression, and thus may be substantially pure
  • an isolated NAP conjugate is unaccompanied by at least some of the material with which it is normally associated in its natural (unpunfied) state, preferably constituting at least about 0 5%, more preferably at least about 5% by weight or more of the total protein in a given sample
  • a substantially pure protein comprises at least about 75% by weight or more of the total protein, with at least about 80% or more being preferred, and at least about 90% or more being particularly preferred NAP conjugates may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample.
  • Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC chromatography, gel filtration, and chromatofocusing. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, NY (1982). The degree of purification necessary will vary depending on the use of the NAP conjugate. In some instances no purification will be necessary.
  • the invention provides for NAP conjugates that are either in solution, optionally purified or isolated, or contained within host cells. Once expressed and purified if necessary, the NAP conjugates are useful in a number of applications, including in vitro and ex vivo screening techniques.
  • in vitro and ex vivo embodiments of the present inventive method have utility in a number of fields of study.
  • the present invention has utility in diagnostic assays and can be employed for research in numerous disciplines, including, but not limited to, clinical pharmacology, functional genomics, pharamcogenomics, agricultural chemicals, environmental safety assessment, chemical sensor, nutrient biology, cosmetic research, and enzymology.
  • the NAP conjugates are used in in vitro screening techniques.
  • the NAP conjugates are made and screened for binding and/or modulation of bioactivites of target molecules.
  • One of the strengths of the present invention is to allow the identification of target molecules that bind to the candidate proteins. As is more fully outlined below, this has a wide variety of applications, including elucidating members of a signaling pathway, elucidating the binding partners of a drug or other compound of interest, etc.
  • target molecules are used in assays with target molecules.
  • target molecules or grammatical equivalents herein is meant a molecule for which an interaction is sought; this term will be generally understood by those in the art.
  • Target molecules include both biological and non-biological targets.
  • Biological targets refer to any defined and non-defined biological particles, such as macromolecular complexes, including viruses, cells, tissues and combinations, that are produced as a result of biological reactions in cells.
  • Non-biological targets refer to molecules or structure that are made outside of cells as a result of either human or non-human activity.
  • the inventive library can also be applied to both chemically defined targets and chemically non-defined targets.
  • “Chemically defined targets” refer to those targets with known chemical nature and/or composition; “chemically non-defined targets” refer to targets that have either unknown or partially known chemical nature/composition.
  • suitable target molecules encompass a wide variety of different classes, including, but not limited to, cells, viruses, proteins (particularly including enzymes, cell-surface receptors, ion channels, and transcription factors, and proteins produced by disease-causing genes or expressed during disease states), carbohydrates, fatty acids and lipids, nucleic acids, chemical moieties such as small molecules, agricultural chemicals, drugs, ions (particularly metal ions), polymers and other biomaterials.
  • binding to polymers both naturally occurring and synthetic), or other biomaterials, may be done using the methods and compositions of the invention.
  • the target is a nucleic acid sequence and the desired candidate protein has the ability to bind to the nucleic acid sequence.
  • the present invention is well suited for identification of DNA binding peptides and their coding sequences, as well as the target nucleic acids that are recognized and bound by the DNA binding peptides. It is known that DNA-protein interactions play important roles in controlling gene expression and chromosomal structure, thereby determining the overall genetic program in a given cell. It is estimated that only 5% of the human genome is involved in coding proteins. Thus, the remaining 95% may be sites with which DNA binding proteins interact, thereby controlling a variety of genetic programs such as regulation of gene expression.
  • the population of target DNA segments can be, for instance, random DNA, fragmented genomic DNA, degenerate sequences, or DNA sequences of various primary, secondary or tertiary structures.
  • the specificity of the DNA binding peptide-substrate binding can be varied by changing the length of the recognition sequence of the target DNA, if desired. Binding of the potential DNA binding peptide to a member of the population of target DNA segments is detected, and further study of the particular DNA recognition sequence bound by the DNA binding peptide can be performed.
  • the population of DNA segments can be bound to, for example, beads or constructed as DNA arrays on microchips.
  • the present invention provides methods for creating a map of DNA binding sequences and DNA binding proteins according to their relative positions, to provide chromosome maps annotated with proteins and sequences. A database comprising such information would then allow for correlating gene expression profiles, disease phenotype, pharmacogenomic data, and the like.
  • the NAP conjugates are used in screens to assay binding to target molecules and/or to screen candidate agents for the ability to modulate the activity of the target molecule.
  • screens are designed to first find candidate proteins that can bind to target molecules, and then these proteins are used in assays that evaluate the ability of the candidate protein to modulate the target's bioactivity.
  • assays there are a number of different assays which may be run; binding assays and activity assays. As will be appreciated by those in the art, these assays may be run in a variety of configurations, including both solution-based assays and utilizing support-based systems.
  • the assays comprise combining the NAP conjugates of the invention and a target molecule, and determining the binding of the candidate protein of the NAP conjugate to the target molecule.
  • libraries of NAP conjugates e.g. comprising a library of different candidate proteins
  • the detection of the interactions of candidate ligands with candidate proteins can be detected using non-denaturing gel electrophoresis.
  • the target ligand is linked to either a primary or secondary label as outlined herein.
  • the labeled target ligand (or libraries of such ligands) is then incubated with a NAP conjugate library and run on a non-denaturing gel as is well known in the art.
  • the visualization of the label allows the excision of the relevant bands followed by isolation of the NAP-conjugate using the techniques outlined herein such as PCR amplification), which can then be verified or used in additional rounds of panning.
  • one of the components of the invention is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.).
  • isolated sample receiving areas e.g. a microtiter plate, an array, etc.
  • the insoluble support may be made of any composition to which the assay component can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening
  • the surface of such supports may be solid or porous and of any convenient shape
  • suitable insoluble supports include microtiter plates, arrays, membranes and beads These are typically made of glass, plastic (e g , polystyrene), polysacchandes, nylon or nitrocellulose, teflon®, etc Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples Alternatively, bead-based assays may be used, particularly with use with fluorescence activated cell sorting (FACS)
  • FACS fluorescence activated cell sorting
  • the NAP conjugates of the invention are arrayed as is generally outlined in U S S N s 09/792,405 and 09/792,630, filed 2/22/01 , both of which are expressly incorporated by reference
  • NAP vectors that also contain capture sequences that will hybridize with capture probes on the surface of a biochip are used, such that the NAP conjugates can be "captured” or “arrayed” on the biochip
  • These protein biochips can then be used in a wide variety of ways, including diagnosis (e g detecting the presence of specific target analytes), screening (looking for target analytes that bind to specific proteins), and smgle-nucleotide polymorphism (SNP) analysis
  • the target analytes can be arrayed on a biochip and the NAP conjugates panned against these biochips
  • the soluble component of the assay be labeled
  • the NAP conjugates can contain a fusion partner comprising a primary or secondary label
  • Preferred embodiments utilize autofluorescent proteins, including, but not limited to, green fluorescent proteins and derivatives from Aqueorea species, Ptil species, and Renilla species
  • the target analytes can be labeled, again using any number of primary or secondary labels as defined herein
  • biochips comprising a substrate with an array of molecules
  • biochip or “array” herein is meant a substrate with a plurality of biomolecules in an array format, the size of the array will depend on the composition and end use of the array
  • the biochips comprise a substrate
  • substrate or “solid support” or other grammatical equivalents herein is meant any material appropriate for the attachment of capture probes and is amenable to at least one detection method. As will be appreciated by those in the art, the number of possible substrates is very large.
  • Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, ceramics, and a variety of other polymers.
  • the substrates allow optical detection and do not themselves appreciably fluoresce.
  • the substrate may be coated with any number of materials, including polymers, such as dextrans, acrylamides, gelatins, agarose, biocompatible substances such as proteins including bovine and other mammalian serum albumin, etc.
  • polymers such as dextrans, acrylamides, gelatins, agarose, biocompatible substances such as proteins including bovine and other mammalian serum albumin, etc.
  • Preferred substrates include silicon, glass, polystyrene and other plastics and acrylics.
  • the substrate is flat (planar), although as will be appreciated by those in the art, other configurations of substrates may be used as well, including the placement of the probes on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
  • array herein is meant a plurality of capture probes in an array format; the size of the array will depend on the composition and end use of the array. Arrays containing from about 2 different capture probes to many thousands can be made. Generally, the array will comprise from two to as many as 100,000 or more, depending on the size of the pads, as well as the end use of the array. Preferred ranges are from about 2 to about 10,000, with from about 5 to about 1000 being preferred, and from about 10 to about 100 being particularly preferred.
  • compositions of the invention may not be in array format; that is, for some embodiments, compositions comprising a single capture probe may be made as well.
  • multiple substrates may be used, either of different or identical compositions.
  • large arrays may comprise a plurality of smaller substrates.
  • the biochip substrates comprise an array of capture probes.
  • capture probes herein is meant nucleic acids (attached either directly or indirectly to the substrate as is more fully outlined below ) that are used to bind, e.g. hybridize, the NAP conjugates of the invention.
  • Capture probes comprise nucleic acids as defined herein.
  • Capture probes are designed to be substantially complementary to capture sequences of the vectors, as is described below, such that hybridization of the capture sequence and the capture probes of the present invention occurs. As outlined below, this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the capture sequences and the capture probes of the present invention. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary sequence. Thus, by “substantially complementary” herein is meant that the probes are sufficiently complementary to the capture sequences to hybridize under normal reaction conditions.
  • Nucleic acid arrays are known in the art, and include, but are not limited to, those made using photolithography techniques (Affymetrix GeneChipTM), spotting techniques (Synteni and others), printing techniques (Hewlett Packard and Rosetta), three dimensional "gel pad” arrays (U.S. Patent No. 5,552,270), nucleic acid arrays on electrodes and other metal surfaces (WO 98/20162; WO 98/12430; WO 99/57317; and WO 01/07665) microsphere arrays (U.S. Patent No. 6,023,540; WO 00/16101 ; WO 99/67641; and WO 00/39587), arrays made using functionalized materials (see PhotoLinkTM technology from SurModics); all of which are expressly incorporated by reference.
  • Photolithography techniques Affymetrix GeneChipTM
  • spotting techniques Synteni and others
  • printing techniques Hewlett Packard and Rosetta
  • three dimensional "gel pad” arrays U.S. Patent
  • the capture probes or candidate ligands can be attached either directly to the substrate, or indirectly, through the use of polymers or through the use of microspheres.
  • Preferred methods of binding to the supports include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the use of labeled components (e.g. the assay component is biotinylated and the surface comprises strepavidin, etc.) the synthesis of the target on the surface, etc.
  • labeled components e.g. the assay component is biotinylated and the surface comprises strepavidin, etc.
  • excess unbound material is removed by suitable methods including, for example, chemical, physical, and biological separation techniques.
  • the sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
  • BSA bovine serum albumin
  • the ligands are attached to silica surfaces such as glass slides or glass beads, using techniques sometimes referred to as "small molecule printing” (SMP) as outlined in MacBeath et al., J. Am. Chem. Soc. 121(34):7967 (1999); Macbeath et al., Science 289:1760; Hergenrother et al., J. Am. Chem. Soc. 122(32):7849 (2000), all of which are expressly incorporated herein by reference. This generally relies on a maleimide derivatized glass slides. Thiol-containing compounds readily attach to the surface upon printing.
  • a preferred method of this embodiment uses traditional "split and mix" combinatorial synthesis of small molecule ligands, using beads for example.
  • the beads can be "tagged” or “encoded” during synthesis.
  • the attachment of the ligands to the beads is labile in some way, frequently either chemically cleavable or photocleavable.
  • the candidate targets or the NAP conjugates can be attached to beads and screened against the other component.
  • the beads can be encoded or tagged using traditional methods, such as the incorporation of dyes or other labels, or nucleic acid "tags".
  • the beads can be encoded on the basis of physical parameters, such as bead size or composition, or combinations.
  • target analytes are attached to glass surfaces or beads, wherein a single glass bead size corresponds to a homogeneous population of molecules. Pools of different sized beads containing different targets are pooled, and the binding assays using the NAP conjugates are run. The beads are then sorted on the basis of size using any number of sizing techniques (meshing, filtering, etc.), and beads containing NAP conjugates can then identified, the NAP conjugates eluted, amplified, validated, etc.
  • multiple targets could be attached to the same size beads, and "hits" could then be deconvoluted later.
  • different coding schemes for beads can be used. For example, beads with magnetic cores in different sizes can be used, or dyes could be incorporated, etc.
  • the target molecule is bound to the support, and a NAP conjugate is added to the assay.
  • the NAP conjugate is bound to the support and the target molecule is added.
  • Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. Determination of the binding of the target and the candidate protein is done using a wide variety of assays, including, but not limited to labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, the detection of labels, functional assays (phosphorylation assays, etc.) and the like.
  • the determination of the binding of the candidate protein to the target molecule may be done in a number of ways.
  • one of the components preferably the soluble one, is labeled, and binding determined directly by detection of the label. For example, this may be done by attaching the NAP conjugate to a solid support, adding a labeled target molecule (for example a target molecule comprising a fluorescent label), removing excess reagent, and determining whether the label is present on the solid support.
  • a labeled target molecule for example a target molecule comprising a fluorescent label
  • This system may also be run in reverse, with the target (or a library of targets) being bound to the support and a NAP conjugate, preferably comprising a primary or secondary label, is added.
  • NAP conjugates comprising fusions with GFP or a variant may be particularly useful.
  • Various blocking and washing steps may be utilized as is known in the art.
  • the solid support is in an array format; that is, a biochip is used which comprises one or more libraries of either candidate agents, targets (including ligands such as small molecules) or NAP conjugates attached to the array.
  • a biochip which comprises one or more libraries of either candidate agents, targets (including ligands such as small molecules) or NAP conjugates attached to the array.
  • targets including ligands such as small molecules
  • NAP conjugates attached to the array.
  • the nucleic acid targets are on the array and the NAP conjugates are added.
  • protein biochips of libraries of target proteins can be used, with labeled NAP conjugates added.
  • the NAP conjugates can be attached to the chip, either through the nucleic acid or through the protein components of the system.
  • only one of the components is labeled; alternatively, more than one component may be labeled with different labels.
  • the binding of the candidate protein is determined through the use of competitive binding assays.
  • the competitor is a binding moiety known to bind to the target molecule such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding as between the target and the binding moiety, with the binding moiety displacing the target.
  • a preferred utility of the invention is to determine the components to which a drug will bind. That is, there are many drugs for which the targets upon which they act are unknown, or only partially known.
  • NAP conjugates comprising a library of cDNA expression products from the cell type on which the drug acts
  • the elucidation of the proteins to which the drug binds may be elucidated.
  • these newly identified proteins can be used in additional drug screens, as a tool for counterscreens, or to profile chemically induced events.
  • Positive controls and negative controls may be used in the assays.
  • Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound. Similarly, ELISA techniques are generally preferred.
  • reagents may be included in the screening assays. These include reagents such as, but not limited to, salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, co-factors such as cAMP, ATP, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
  • reagents such as, but not limited to, salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions.
  • reagents that otherwise improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, anti-microbial agents, co-factors such as cAMP, ATP, etc.
  • methods for screening for a candidate protein capable of modulating the activity of the target molecule comprise the steps of adding a NAP conjugate to a sample of the target, as above, and determining an alteration in the biological activity of the target.
  • “Modulation” or “alteration” in this context includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present.
  • the candidate protein should both bind to the target (although this may not be necessary), and alter its biological or biochemical activity as defined herein.
  • the methods include both in vitro screening methods, as are generally outlined above, and ex vivo screening of cells for alterations in the presence, distribution, activity or amount of the target.
  • a candidate peptide can be identified that does not interfere with target activity, which can be useful in determining drug-drug interactions.
  • the methods comprise combining a target molecule and preferably a library of NAP conjugates and evaluating the effect on the target molecule's bioactivity. This can be done in a wide variety of ways, as will be appreciated by those in the art.
  • the NAP conjugate is retrieved to allow identification of the candidate protein. Retrieval of the NAP conjugate can be done in a wide variety of ways, as will be appreciated by those in the art and will also depend on the type and configuration of the system being used.
  • a rescue tag or "retrieval property” is used.
  • a "retrieval property” is a property that enables isolation of the fusion enzyme when bound to the target.
  • the target can be constructed such that it is associated with biotin, which enables isolation of the target-bound fusion enzyme complexes using an affinity column coated with streptavidin.
  • the target can be attached to magnetic beads, which can be collected and separated from non-binding candidate proteins by altering the surrounding magnetic field.
  • the NAP conjugate may comprise the rescue tag.
  • affinity tags may be incorporated into the fusion proteins themselves.
  • the fusion enzyme-nucleic acid molecule complex can be also recovered by immunoprecipitation.
  • rescue tags may comprise unique vector sequences that can be used to PCR amplify the nucleic acid encoding the candidate protein. In the latter embodiment, it may not be necessary to break the covalent attachment of the nucleic acid and the protein, if PCR sequences outside of this region (that do not span this region) are used.
  • the covalent linkage between the fusion enzyme and its coding nucleic acid molecule can be severed using, for instance, nuclease-free proteases, the addition of non-specific nucleic acid, or any other conditions that preferentially digest proteins and not nucleic acids.
  • the nucleic acid molecules are purified using any suitable methods, such as those methods known in the art, and are then available for further amplification, sequencing or evolution of the nucleic acid sequence encoding the desired candidate protein. Suitable amplification techniques include all forms of PCR, OLA, SDA, NASBA, TMA, Q- ⁇ R, etc. Subsequent use of the information of the "hit" is discussed below.
  • the NAP conjugates are used in ex vivo screening techniques.
  • the expression vectors of the invention are introduced into host cells to screen for candidate proteins with a desired property, e.g., capable of altering the phenotype of a cell.
  • An advantage of the present inventive method is that screening of the fusion enzyme library can be accomplished intracellularly.
  • screening candidate proteins within their natural environment, as opposed to lysing the cell to screen in vitro.
  • variant peptides are displayed in their native conformation and are screened in the presence of other possibly interfering or enhancing cellular agents. Accordingly, screening intracellularly provides a more accurate picture of the actual activity of the candidate protein and, therefore, is more predictive of the activity of the peptide ex vivo or in vivo.
  • the effect of the candidate protein on cellular physiology can be observed.
  • Ex vivo and/or in vivo screening can be done in several ways.
  • the target need not be known; rather, cells containing the expression vectors of the invention are screened for changes in phenotype. Cells exhibiting an altered phenotype are isolated, and the target to which the NAP conjugate bound is identified as outlined below, although as will be appreciated by those in the art and outlined herein, it is also possible to bind the fusion polypeptide and the target prior to forming the NAP conjugate.
  • the target may be added exogeneously to the cell and screening for binding and/or modulation of target activity is done.
  • the target should be able to penetrate the membrane, by, for instance, direct penetration or via membrane transporting proteins, or by fusions with transport moieties such as lipid moieties or HIV-tat, described below.
  • NAP conjugates within the cells prior to screening, although this is not required. That is, the attachment of the NAM fusion enzyme to the EAS may occur at any time during the screening, either before, during or after, as long as the conditions are such that the attachment occurs prior to mixing of cells or cell lysates containing different fusion nucleic acids.
  • the type of cells used in this embodiment can vary widely. Basically, any procaryotic cells can be used.
  • the host cells can be singular cells, or can be present in a population of cells, such as in a cell culture. As is more fully described below, a screen will be set up such that the cells exhibit a selectable phenotype in the presence of a candidate protein.
  • the cells may be genetically engineered, that is, contain exogenous nucleic acid, for example, to contain target molecules.
  • a first plurality of cells is screened. That is, the cells into which the expression vectors are introduced are screened for an altered phenotype.
  • the effect of the candidate protein is seen in the same cells in which it is made; i.e. an autocrine effect.
  • a “plurality of cells” herein is meant roughly from about 10 3 cells to 10 8 or 10 9 , with from 10 6 to 10 8 being preferred.
  • This plurality of cells comprises a cellular library, wherein generally each cell within the library contains a member of the NAP conjugate molecular library, i.e. a different candidate protein, although as will be appreciated by those in the art, some cells within the library may not contain an expression vector and some may contain more than one.
  • the expression vectors are introduced into a first plurality of cells, and the effect of the candidate proteins is screened in a second or third plurality of cells, different from the first plurality of cells, i.e. generally a different cell type. That is, the effect of the candidate protein is due to an extracellular effect on a second cell; i.e. an endocrine or paracrine effect.
  • the first plurality of cells may be grown in or on one media, and the media is allowed to touch a second plurality of cells, and the effect measured. Alternatively, there may be direct contact between the cells. Thus, "contacting" is functional contact, and includes both direct and indirect.
  • the first plurality of cells may or may not be screened.
  • the cells are treated to conditions suitable for the expression of the fusion nucleic acids (for example, when inducible promoters are used), to produce the candidate proteins.
  • a first plurality of cells is used for library construction and a second plurality of cells is used for the expression of the library.
  • the first plurality of cells may be transformed with plasmid expression vectors comprising fusion nucleic acids comprising a T7 promoter operably linked to a nucleic acid encoding a NAM enzyme and a candidate protein and a EAS.
  • the T7 promoter is located on either a pET-24a(+) vector or a GatewayTM vector as described above. This method is preferred when the nucleic acid encoding the candidate proteins is from a eucaryotic organism, such as yeast, mammals, humans, etc.
  • the nucleic acid may be genomic DNA or cDNA.
  • Suitable cells include Top10 cells (Invitrogen), DH10B (Invitrogen) and DH5 ⁇ (Invitrogen).
  • a second plurality of cells is transformed using the library DNA obtained from the first plurality of cells for expression and panning.
  • Suitable cells include BL21(DE3) (Novagen). Two advantages associated with this approach are: 1) preventing the expression of toxic genes during the library construction phase; and 2) the library is more stable and a greater number of genes may be cloned, expressed and panned.
  • the methods of the present invention preferably comprise introducing a molecular library of fusion nucleic acids or expression vectors into a plurality of cells, thereby creating a cellular library.
  • two or more of the nucleic acids comprises a different nucleotide sequence encoding a different candidate protein
  • the plurality of cells is then screened, as is more fully outlined below, for a cell exhibiting an altered phenotype
  • the altered phenotype is due to the presence of a candidate protein
  • altered phenotype or “changed physiology” or other grammatical equivalents herein is meant that some detectable and/or measurable characteristic of the cell is altered in some way Accordingly, any change which may be observed, detected, or measured may be the basis of the screening methods herein Suitable changes include, but are not limited to temperature sensitivity, resistance or susceptibility to a given agent, such as an antibiotic, colony size, etc
  • capable of altering the phenotype herein is meant that the candidate protein can change the phenotype of the cell in some detectable and/or measurable way
  • the altered phenotype may be detected in a wide variety of ways, as is described more fully below, and will generally depend and correspond to the phenotype that is being changed Generally, the changed phenotype is detected using, for example microscopic analysis of cell morphology, standard cell viability assays, including both increased cell death and increased cell viability, for example, cells that are now resistant to synthetic toxins or antibiotics, standard labeling assays such as fluorometric indicator assays for the presence or level of a particular cell or molecule, including FACS or other dye staining techniques, biochemical detection of the expression of target compounds after killing the cells, etc
  • compositions and methods of the invention are used to detect protein- protein interactions, similar to the use of a two-hybrid screen This can be done in a variety of ways and in a variety of formats As will be appreciated by those in the art, this embodiment and others outlined herein can be run as a "one dimensional” analysis or "multidimensional” analysis That is, one NAP conjugate library can be run against a single target or against a library of targets Alternatively, more than one NAP conjugate library can be run against each other
  • compositions and methods of the invention are used in protein drug discovery, particularly for protein drugs that interact with targets on cell surfaces
  • compositions and methods of the invention are used to discover DNA or nucleic acid binding proteins, using nucleic acids as the targets
  • the libraries are pre-separated into sublibranes that are employed to identify specific enzymatic components within each sublibrary
  • target analytes or ligands that are substrates e g are modified by enzymes to release or generate a specific signal which may be detected, preferably optically (e g spectophotomet ⁇ cally, fluorescently, etc )
  • phosphatases may be visualized by employing organophosphates, which when hydrolyzed release p-nitrophenol, which is monitored at 350 nm
  • the sublibranes are generated by diluting standard sized libraries (e g 10 7 ) and then splitting the library into sublibrary pools Each individual pool can then be independently transformed into host cells such as bacteria, amplified and isolated Each pool is then amplified, isolated and lysed to yield cell lysates The ligand substrates are then added to the lysates, and "hits" identified optically and collected This process may optionally be reiterated, followed by transformation of the well contents into bacterial cells and plated Individual colonies are picked, the plasmids in vitro translated and the products treated with the ligand substrates All active clones are then identified and characterized as outlined herein
  • compositions and methods of the invention are used to screen for NAM enzymes with decreased toxicity for the host cells
  • Rep proteins of the invention can be toxic to some host cells
  • the present inventive methods can be used to identify or generate Rep proteins with decreased toxicity
  • Rep variants or, in an alternative, random peptides are used in the present inventive conjugates to observe cell toxicity and binding affinity to an EAS
  • the present inventive methods can also be utilized to identify novel or improved EASs for use in the present inventive expression vectors
  • An EAS for a particular NAM enzyme of interest can also be identified using the present inventive method
  • Formation of covalent structure of NAM enzyme and EAS can determined using suitable methods that are present in the art, e g those described in U S patent 5545529
  • the candidate NAM enzyme can be expressed using a variety of hosts, such as bacteria or mammalian cells
  • candidate DNA sequences such a library of fragments obtained from the genome from which the NAM enzyme is cloned
  • Contacts between the NAM enzyme and with the library of DNA fragments under appropriate conditions (such as inclusion of cofactors) allow for the formation of covalent NAM enzyme-DNA conjugates
  • the mixture can then be separated using a variety of techniques
  • the isolated bound nucleic acid sequences can then be identified and sequenced These sequences can be tested further via a variety of mutagenesis techniques
  • the confirmed sequence motif can then be used an EAS
  • compositions and methods of the invention are used in pharmacogenetic studies For example, by building libraries from individuals with different phenotypes and testing them against targets, differential binding profiles can be generated.
  • a preferred embodiment utilizes differential binding profiles of NAP conjugates to targets to elucidate disease genes, SNPs or proteins.
  • this process is outlined as follows.
  • the first primary screen is run, using a tagged compound of interest panned against a library of NAP conjugates. This tagged compound is used to isolate all candidate proteins that bind to it. By decoding the cDNA of the isolated candidates, all possible candidates for the secondary screen are identified.
  • the secondary screen then is initiated by directly or indirectly covalently linking the primary candidate hits to a solid support, using any number of known techniques such as those outlined herein.
  • the linkage technique should not interfere with the binding site of the original tagged compound, and should maximize the ability of the protein to interact with other proteins.
  • a variety of different linkages and/or linkage sites are used, and may include the additional use of linkers as outlined herein.
  • the secondary screen proceeds with the incubation of the array of attached candidate proteins with the original compound of interest, preferably in an untagged form, in the presence of a NAP conjugate library.
  • the NAP conjugate library may be first incubated with the candidate protein linked to a solid support (in the absence of the ligand), and all entities that are not retained on the solid support are used in the screen. Subsequent isolation and decoding of the cDNA of the candidate proteins that bind the protein-ligand complex thus identifies additional interactions mediated by the ligand.
  • the cell is isolated from the plurality which do not have altered phenotypes. This may be done in any number of ways, as is known in the art, and will in some instances depend on the assay or screen. Suitable isolation techniques include, but are not limited to, expression of a "survival" protein, induced expression of a cell surface protein or other molecule that can be rendered fluorescent or taggable for physical isolation, expression of an enzyme that changes a non-fluorescent molecule to a fluorescent one, overgrowth against a background of no or slow growth, death of cells and isolation of DNA or other cell vitality indicator dyes, etc
  • the NAP conjugate is isolated from the positive cell This may be done in a number of ways
  • primers complementary to DNA regions common to the NAP constructs, or to specific components of the library such as a rescue sequence, defined above are used to "rescue" the unique candidate protein sequence
  • the candidate protein is isolated using a rescue sequence
  • rescue sequences comprising epitope tags or purification sequences may be used to pull out the candidate protein, using immunoprecipitation or affinity columns In some instances, as is outlined below, this may also pull out the primary target molecule, if there is a sufficiently strong binding interaction between the candidate protein and the target molecule
  • the peptide may be detected using mass spectroscopy Once rescued, the sequence of the candidate protein and fusion nucleic acid can be determined This information can then be used in a number of ways, i e , genomic databases
  • the candidate protein is resynthesized and remtroduced into the target cells, to verify the effect
  • recombinant methods e g by transforming naive cells with the expression vector (or modified versions, e g with the candidate protein no longer part of a fusion), or alternatively using fusions to the HIV-1 Tat protein, and analogs and related proteins, which allows very high uptake into target cells
  • Fawell et al PNAS USA 91 664 (1994), Frankel et al , Cell 55 1189 (1988), Savion et al , J Biol Chem 256 1149 (1981), Derossi et al , J Biol Chem 269 10444 (1994), and Bald
  • the process may be used reiteratively That is, the sequence of a candidate protein is used to generate more candidate proteins
  • the sequence of the protein may be the basis of a second round of (biased) randomization, to develop agents with increased or altered activities Alternatively, the second round of randomization may change the affinity of the agent
  • the candidate protein is a random peptide
  • the methods of using the present inventive library can involve many rounds of screenings in order to identify a nucleic acid of interest.
  • the method can be repeated using a different target. Multiple libraries can be screened in parallel or sequentially and/or in combination to ensure accurate results. In addition, the method can be repeated to map pathways or metabolic processes by including an identified candidate protein as a target in subsequent rounds of screening.
  • the candidate protein is used to identify target molecules, i.e. the molecules with which the candidate protein interacts.
  • target molecules i.e. the molecules with which the candidate protein interacts.
  • the candidate protein is used to pull out target molecules.
  • the target molecules are proteins
  • the use of epitope tags or purification sequences can allow the purification of primary target molecules via biochemical means (co-immunoprecipitation, affinity columns, etc.).
  • the peptide when expressed in bacteria and purified, can be used as a probe against a bacterial cDNA expression library made from mRNA of the target cell type.
  • peptides can be used as "bait" in either yeast or mammalian two or three hybrid systems. Such interaction cloning approaches have been very useful to isolate DNA-binding proteins and other interacting protein components.
  • the peptide(s) can be combined with other pharmacologic activators to study the epistatic relationships of signal transduction pathways in question. It is also possible to synthetically prepare labeled peptides and use it to screen a cDNA library expressed in bacteriophage for those cDNAs which bind the peptide.
  • secondary target molecules may be identified in the same manner, using the primary target as the "bait". In this manner, signaling pathways may be elucidated. Similarly, protein agents specific for secondary target molecules may also be discovered, to allow a number of protein agents to act on a single pathway, for example for combination therapies.
  • the methods and compositions of the invention can be performed using a robotic system.
  • Many systems are generally directed to the use of 96 (or more) well microtiter plates, but as will be appreciated by those in the art, any number of different plates or configurations may be used.
  • any or all of the steps outlined herein may be automated; thus, for example, the systems may be completely or partially automated.
  • a wide variety of automatic components can be used to perform the present inventive method or produce the present inventive compositions, including, but not limited to, one or more robotic arms; plate handlers for the positioning of microplates; automated lid handlers to remove and replace lids for wells on non-cross contamination plates; tip assemblies for sample distribution with disposable tips; washable tip assemblies for sample distribution; 96 well loading blocks; cooled reagent racks; microtiter plate pipette positions (optionally cooled); stacking towers for plates and tips; and computer systems.
  • Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism-handling including high throughput pipetting to perform all steps of screening applications.
  • This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers; retrieving, and discarding of pipet tips; and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration.
  • These manipulations are cross-contamination-free liquid, particle, cell, and organism transfers.
  • This instrument performs automated replication of microplate samples to filters, membranes, and/or daughter plates, high- density transfers, full-plate serial dilutions, and high capacity operation.
  • chemically derivatized particles, plates, tubes, magnetic particle, or other solid phase matrix with specificity to the assay components are used.
  • the binding surfaces of microplates, tubes or any solid phase matrices include non-polar surfaces, highly polar surfaces, modified dextran coating to promote covalent binding, antibody coating, affinity media to bind fusion proteins or peptides, surface-fixed proteins such as recombinant protein A or G, nucleotide resins or coatings, and other affinity matrix are useful in this invention.
  • platforms for multi-well plates, multi-tubes, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity.
  • This modular platform includes a variable speed orbital shaker, electroporator, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.
  • thermocycler and thermoregulating systems are used for stabilizing the temperature of the heat exchangers such as controlled blocks or platforms to provide accurate temperature control of incubating samples from 4 C to 100°C.
  • interchangeable pipet heads with single or multiple magnetic probes, affinity probes, or pipetters robotically manipulate the liquid, particles, cells, and organisms.
  • Multi-well or multi-tube magnetic separators or platforms manipulate liquid, particles, cells, and organisms in single or multiple sample formats.
  • the instrumentation will include a detector, which can be a wide variety of different detectors, depending on the labels and assay.
  • useful detectors include a microscope(s) with multiple channels of fluorescence; plate readers to provide fluorescent, ultraviolet and visible spectrophotometric detection with single and dual wavelength endpoint and kinetics capability, fluorescence resonance energy transfer (FRET), luminescence, quenching, two-photon excitation, and intensity redistribution; CCD cameras to capture and transform data and images into quantifiable formats; and a computer workstation. These will enable the monitoring of the size, growth and phenotypic expression of specific markers on cells, tissues, and organisms; target validation; lead optimization; data analysis, mining, organization, and integration of the high-throughput screens with the public and proprietary databases.
  • FRET fluorescence resonance energy transfer
  • Flow cytometry or capillary electrophoresis formats can be used for individual capture of magnetic and other beads, particles, cells, and organisms.
  • the flexible hardware and software allow instrument adaptability for multiple applications.
  • the software program modules allow creation, modification, and running of methods.
  • the system diagnostic modules allow instrument alignment, correct connections, and motor operations.
  • the customized tools, labware, and liquid, particle, cell and organism transfer patterns allow different applications to be performed.
  • the database allows method and parameter storage. Robotic and computer interfaces allow communication between instruments.
  • the robotic workstation includes one or more heating or cooling components.
  • either cooling or heating may be required, which can be done using any number of known heating and cooling systems, including Peltier systems.
  • the robotic apparatus includes a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus.
  • a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus.
  • input/output devices e.g., keyboard, mouse, monitor, printer, etc.
  • the above-described methods of screening a pool of fusion enzyme-nucleic acid molecule complexes for a nucleic acid encoding a desired candidate protein are merely based on the desired target property of the candidate protein.
  • the sequence or structure of the candidate proteins does not need to be known.
  • a significant advantage of the present invention is that no prior information about the candidate protein is needed during the screening, so long as the product of the identified coding nucleic acid sequence has biological activity, such as specific association with a targeted chemical or structural moiety.
  • the identified nucleic acid molecule then can be used for understanding cellular processes as a result of the candidate protein's interaction with the target and, possibly, any subsequent therapeutic or toxic activity.
  • pQE82L vectors (Qiagen) were used to construct a vector comprising PCD302 and an EAS.
  • the pPCD302/EAS vector comprises a phage T5 promoter, two Lac operators, a bacterial ribosome binding site (RBSII), an MRGS-His epitope at the N-terminus, multiple cloning site, various stop codons and transcriptional terminatiors and a lacl q repressor gene, a ColE1 origin of replication and an ampicillin gene.
  • Chemically competent Top 10 cells were transformed according to the manufacturer's instructions as follows: DNA was added to the competent cells in an Eppendorf tube on ice for 30 min incubation. After a brief heat shock at 42°C for 30-45 seconds, SOC (a rich medium, 250 ⁇ l) was added and the competent cells were incubated at 37°C with shaking for an hour. The transformed cells were spread onto ampillicin (mp)-LB plates for colony selection.
  • DNA was added to the competent cells in an Eppendorf tube on ice for 30 min incubation. After a brief heat shock at 42°C for 30-45 seconds, SOC (a rich medium, 250 ⁇ l) was added and the competent cells were incubated at 37°C with shaking for an hour. The transformed cells were spread onto ampillicin (mp)-LB plates for colony selection.
  • SOC a rich medium, 250 ⁇ l
  • the cells were transformed with:
  • PCD302Y156F/165 base pair (bp) EAS This is a control plasmid used to determine the effect of the linkage between PCD302 and the EAS on bacterial growth.
  • This plasmid contains a mutation in the PCDeow sequence such that the PCD302 may bind to the EAX, but not form a covalent linkage.
  • a single colony from a fresh Amp-LB plate was picked and incubated in 5 ml CG media (50 ug/ml Amp) in a Falcon tube at 37°C for approximately 15 hours or 20°C for approximately 23 hours and the OD 600 determined.
  • PCD302/EAS inhibits the growth of the transformed cells. This effect was more pronounced at 20°C than at 37°. The growth of cells transformed with the control, PCD302Y156F/EAS was not affected, indicating that this effect is directly due to the PCD302- EAS linkage. Thus, control of PCD302 background expression is critical for minimizing spontaneous PCD or EAS mutation.
  • the inhibitory effects of PCD302/EAS could be minimized by selecting a single colony from a fresh Amp-LB plate and incubated it in 5 ml CG media (50 ug/ml Ap and 2% glucose) in a Falcon tube at 37°C overnight with shaking (the medium could be 2YT, LB or CG).
  • PCD302 Based on our observation that adding 1 to 2% glucose to the culture media reduces the background expression of PCD302, a new generation of vectors were constructed such that the PCD302 gene would not be expressed during the construction of bacterial libraries. In the new constructs, the PCD302 ils placed under control of the T7 promoter.
  • the new vectors are summarized below:
  • pET-24a(+) based expression vectors were used to construct a vector comprising PCD302 and an EAS (see Figure 54).
  • the PCD302 gene was inserted into the Hindlll site, such that expression of the PCD302 gene is under control of the T7 promoter.
  • pET-24a(+) is derived from pBR322 and thus contains the pMB1 origin of replication.
  • An EAS sequence 165 base pairs or less, is inserted upstream of the Sphl site, but the EAS also may be inserted upstream, downstream, inside or outside of the transcriptional unit of PCD302.
  • pBAD/Myc-His vectors were used to construct a vector comprising PCD302 (see Figure 56).
  • the PCD302 gene can be inserted into the Hindlll site, such that the expression of PCD302 is under the control of the araBAD promoter.
  • pBAD is derived from pBR322 and thus contains the pMB1 origin of replication.
  • An EAS sequence 165 base pairs or less, may be inserted upstream, downstream, inside or outside of the transcriptional unit of PCD302.
  • the advantage of the pBAD vector is tight control of the background expression of PCD302.
  • a sequence from the Gateway vector (InvitrogenTM) was used to modify the pQE82L based expression vector (see Figure 53), to make the pQE82L vectors compatible for GatewayTM recombination technoloy.
  • the GatewayTM transfer cassette was inserted into the SnaBI site at the 3' end of PCD302 (see insert, Figure 60A). This modified vector can be used for the GatewayTM transfer of cDNAs from a variety of commercially available GatewayTM mammalian cDNA libraries.
  • MBP Maltose Binding Protein
  • vector constructs comprising PCD302 fused upstream to either the maltose binding protein or FKBP were made.
  • the resulting constructs are shown in Figures 57A and 57B.
  • Bacterial genomic DNA was isolated using the Qiagen genomic DNA isolation kit and fragmented into 0.8 to 2 kb fragments using a Hydroshear GeneMachine .
  • the sheared ends of the DNA fragments were treated with mung bean nuclease and T4 polynucleotide kinase (T4 PNK) to generate blunt ends for cloning.
  • T4 PNK T4 polynucleotide kinase
  • Other methods of generating blunt ends include treating the sheared fragments witheither T4 DNA polymerase plus T4 PNK, or Klenow plus T4 PNK.
  • the DNA was precipitated and purified with a Qiagen PCR purification kit.
  • the purified genomic DNA inserts were then ligated with SnaBI digested PQE82L/PCD302/EAS.
  • the resulting pQE82L/PCD302-genomic insert/EAS constructs were then used to transform an electrically competent host cell, such as DH10B, BL21 , etc., via electroporation.
  • the transformed cells were plated out onto LB plates containing ampicillin +1% glucose.
  • the diversity may statistically include all the genes for fusion expression once.
  • a two step method was used to transfer commercial (ResGen, a subsidiary of Invitrogen) GatewayTM human skeletal muscle CDNA library into the PQE82L vector modified to be compatible with the GatewayTM system.
  • Preliminary analysis of the resultant library showed comparable average cDNA insert size similar to the one from the original cDNA library, i.e., 1.5 kB.
  • the final library diversity is very high.
  • PCD302-MBP Recovery of the PCD302-MBP was measured by mixing cells transformed with PCD302-MBP construct with cells transformed with the PCD302 construct or with cells transformed with a bacterial genomic library. After mixing, the cells were lysed with 50% B-per (Pierce) for 30 minutes and the lysate was cleared by centrifugation at 46,000 g at 4°C for 30 minutes. The lysate was incubated at 4°C for one hour with amylose coated beads to recover PCD302/MBP containing constructs. After extensive washes with phosphate buffered saline (PBS), the bound MBP-DNA complex was eluted with 10% maltose in PBS at room temperature.
  • PBS phosphate buffered saline
  • the DNA elution was treated with 4M guanidine (optional at high temperature) and purified with Qiagen PCR purification kit.
  • the purified plasmid DNA was optionally treated with proteinase K and used to transform competent host cells via electroporation.
  • the resulting colonies were selected on LB-Amp plates and the identity of the plasmids identified using colony-PCR with a pair of MBP specific primiers or a combination of MBP primer and a PCD302 vector primer.
  • the final readout is the DNA agarose gel of the specific PCR products (see Figure 60).
  • the DNA gel results shown in Figure 62 illustrate that greater than 80% of the MBP constaining constructs are recovered using the amylose beads, indicating highly efficient recovery of the PCD302/MBP constructs from the background library.
  • the DNA gels results shown in Figure 63 are from a representative experiment in which the PCD302/MBP construct was mixed with a bacterial genomic library (low diversity) at 10 4 , 10 5 , 10 6 dilutions. As can be seen from the results shown in Figure 63 over 90% of the colonies picked from the 10 4 dilution contained the PCD302/MBP fusion. Over 80% of the colonies selected from the 10 5 dilution contained the PCD302/MBP fusion. Over 30% of the colonies selected from the 10 s dilution contained the PCD302/MBP fusion. Similar results were obtained with the PCD302/FKBP construct.

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Abstract

L'invention concerne des méthodes et des compositions permettant de construire des banques procaryotiques exprimant des protéines candidates, et l'utilisation de ces banques procaryotiques pour identifier lesdites protéines candidates et les acides nucléiques codant pour celles-ci.
PCT/US2001/049058 2000-12-14 2001-12-14 Banques procaryotiques et leurs utilisations WO2002066653A2 (fr)

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WO2003000405A2 (fr) * 2001-02-22 2003-01-03 Xencor, Inc. Biopuces comprenant des conjugues d'acide nucleique/proteine
WO2005030957A1 (fr) * 2003-10-01 2005-04-07 ETH Zürich Procede pour faire evoluer des polypeptides in vitro
US8399618B2 (en) 2004-10-21 2013-03-19 Xencor, Inc. Immunoglobulin insertions, deletions, and substitutions
US9040041B2 (en) 2005-10-03 2015-05-26 Xencor, Inc. Modified FC molecules
US9714282B2 (en) 2003-09-26 2017-07-25 Xencor, Inc. Optimized Fc variants and methods for their generation
US9803023B2 (en) 2004-11-12 2017-10-31 Xencor, Inc. Fc variants with altered binding to FcRn
US10336818B2 (en) 2004-11-12 2019-07-02 Xencor, Inc. Fc variants with altered binding to FcRn
US11401348B2 (en) 2009-09-02 2022-08-02 Xencor, Inc. Heterodimeric Fc variants
US11932685B2 (en) 2007-10-31 2024-03-19 Xencor, Inc. Fc variants with altered binding to FcRn

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US20100311954A1 (en) * 2002-03-01 2010-12-09 Xencor, Inc. Optimized Proteins that Target Ep-CAM
US7662925B2 (en) * 2002-03-01 2010-02-16 Xencor, Inc. Optimized Fc variants and methods for their generation
US8388955B2 (en) * 2003-03-03 2013-03-05 Xencor, Inc. Fc variants
US20090010920A1 (en) * 2003-03-03 2009-01-08 Xencor, Inc. Fc Variants Having Decreased Affinity for FcyRIIb
US8101720B2 (en) 2004-10-21 2012-01-24 Xencor, Inc. Immunoglobulin insertions, deletions and substitutions
US20060134105A1 (en) * 2004-10-21 2006-06-22 Xencor, Inc. IgG immunoglobulin variants with optimized effector function
US8883147B2 (en) 2004-10-21 2014-11-11 Xencor, Inc. Immunoglobulins insertions, deletions, and substitutions
US8802820B2 (en) 2004-11-12 2014-08-12 Xencor, Inc. Fc variants with altered binding to FcRn
US8546543B2 (en) 2004-11-12 2013-10-01 Xencor, Inc. Fc variants that extend antibody half-life
WO2006112885A1 (fr) * 2005-04-14 2006-10-26 The Curators Of The University Of Missouri Systeme et procede pour la prediction d’une variation de sequence et la detection de genie genetique utilisant des motifs de mutation et/ou de substitution documentes codon/acide amine
CA2652434A1 (fr) 2005-07-08 2007-01-18 Xencor, Inc. Proteines optimisees ciblant des ep-cam
US10689643B2 (en) 2011-11-22 2020-06-23 Active Motif, Inc. Targeted transposition for use in epigenetic studies
CN108034616B (zh) * 2018-01-19 2021-03-02 中国农业大学 一种芽孢杆菌的发酵方法

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003000405A2 (fr) * 2001-02-22 2003-01-03 Xencor, Inc. Biopuces comprenant des conjugues d'acide nucleique/proteine
WO2003000405A3 (fr) * 2001-02-22 2003-10-16 Xencor Inc Biopuces comprenant des conjugues d'acide nucleique/proteine
US9714282B2 (en) 2003-09-26 2017-07-25 Xencor, Inc. Optimized Fc variants and methods for their generation
WO2005030957A1 (fr) * 2003-10-01 2005-04-07 ETH Zürich Procede pour faire evoluer des polypeptides in vitro
US8399618B2 (en) 2004-10-21 2013-03-19 Xencor, Inc. Immunoglobulin insertions, deletions, and substitutions
US9803023B2 (en) 2004-11-12 2017-10-31 Xencor, Inc. Fc variants with altered binding to FcRn
US10336818B2 (en) 2004-11-12 2019-07-02 Xencor, Inc. Fc variants with altered binding to FcRn
US11198739B2 (en) 2004-11-12 2021-12-14 Xencor, Inc. Fc variants with altered binding to FcRn
US12215165B2 (en) 2004-11-12 2025-02-04 Xencor, Inc. Fc variants with altered binding to FcRn
US9040041B2 (en) 2005-10-03 2015-05-26 Xencor, Inc. Modified FC molecules
US11932685B2 (en) 2007-10-31 2024-03-19 Xencor, Inc. Fc variants with altered binding to FcRn
US11401348B2 (en) 2009-09-02 2022-08-02 Xencor, Inc. Heterodimeric Fc variants

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